CN102300991A - Plants Having Enhanced Abiotic Stress Tolerance And/or Enhanced Yield-related Traits And A Method For Making The Same - Google Patents
Plants Having Enhanced Abiotic Stress Tolerance And/or Enhanced Yield-related Traits And A Method For Making The SameDownload PDFInfo
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Abstract
Description
Translated fromChinese技术领域technical field
本发明一般地涉及分子生物学领域，涉及通过调节植物中编码alfin样多肽的核酸的表达来增强非生物胁迫耐受性的方法。本发明还涉及具有经调节的alfin样多肽编码核酸表达的植物，所述植物相对于相应的野生型植物或其他对照植物具有增强的非生物胁迫耐受性。本发明还提供了可以用于实施本发明方法的构建体。The present invention relates generally to the field of molecular biology and relates to methods of enhancing abiotic stress tolerance by modulating expression in plants of nucleic acids encoding alfin-like polypeptides. The present invention also relates to plants having modulated expression of an alfin-like polypeptide-encoding nucleic acid, which plants have enhanced abiotic stress tolerance relative to corresponding wild-type plants or other control plants. The invention also provides constructs that can be used to practice the methods of the invention.
此外，本发明一般地涉及分子生物学领域，涉及通过调节植物中编码YRP的核酸的表达来增强植物的非生物胁迫耐受性的方法。本发明还涉及具有经调节的YRP编码核酸表达的植物，所述植物相对于相应的野生型植物或其他对照植物具有增强的非生物胁迫耐受性。本发明还提供了可以用于本发明方法的构建体。Furthermore, the present invention relates generally to the field of molecular biology, and relates to methods for enhancing abiotic stress tolerance in plants by modulating the expression in plants of a nucleic acid encoding YRP. The present invention also relates to plants having modulated expression of a YRP-encoding nucleic acid, which plants have enhanced abiotic stress tolerance relative to corresponding wild-type plants or other control plants. The invention also provides constructs that can be used in the methods of the invention.
此外，本发明一般地涉及分子生物学领域，涉及通过增加植物中编码Brevis Radix-like(BRXL)多肽的核酸序列的表达来增强多种植物产量相关性状的方法。本发明还涉及具有增加的编码BRXL多肽的核酸序列的表达的植物，所述植物相对于对照植物具有增强的产量相关性状。本发明还涉及核酸序列、包含所述核酸序列的核酸构建体、载体和植物。Furthermore, the present invention generally relates to the field of molecular biology, and relates to a method for enhancing various plantyield -related traits by increasing expression in a plant of a nucleic acid sequence encoding aBrevisRadix -like (BRXL) polypeptide. The present invention also relates to plants having increased expression of a nucleic acid sequence encoding a BRXL polypeptide, said plants having enhanced yield-related traits relative to control plants. The invention also relates to nucleic acid sequences, nucleic acid constructs, vectors and plants comprising said nucleic acid sequences.
此外，本发明一般地涉及分子生物学领域，涉及通过调节植物中编码silky-1样多肽的核酸的表达来增强植物的非生物胁迫耐受性的方法。本发明还涉及具有经调节的silky-1样多肽编码核酸表达的植物，所述植物相对于相应的野生型植物或其他对照植物具有增强的非生物胁迫耐受性。本发明还提供了可以用于本发明方法的构建体。Furthermore, the present invention generally relates to the field of molecular biology, and relates to a method for enhancing abiotic stress tolerance in plants by modulating the expression in a plant of a nucleic acid encoding a silky-1-like polypeptide. The present invention also relates to plants having modulated expression of a nucleic acid encoding a silky-1-like polypeptide, which plants have enhanced abiotic stress tolerance relative to corresponding wild-type plants or other control plants. The invention also provides constructs that can be used in the methods of the invention.
此外，本发明一般地涉及分子生物学领域，涉及通过调节植物中编码ARP6(肌动蛋白相关蛋白6)的核酸的表达来改善多种植物生长特征的方法。本发明还涉及具有经调节的ARP编码核酸表达的植物，所述植物相对于相应的野生型植物或其他对照植物具有改善的生长特征。本发明还提供了可以用于本发明方法的构建体。Furthermore, the present invention relates generally to the field of molecular biology and relates to methods for improving various plant growth characteristics by modulating the expression in plants of a nucleic acid encoding ARP6 (Actin-related protein 6). The present invention also relates to plants having modulated expression of an ARP-encoding nucleic acid, which plants have improved growth characteristics relative to corresponding wild-type plants or other control plants. The invention also provides constructs that can be used in the methods of the invention.
此外，本发明一般地涉及分子生物学领域，涉及通过调节植物中编码POP(脯氨酰寡肽酶，Prolyl-oligopeptidase)多肽的核酸的表达来增强植物的产量相关性状的方法。本发明还涉及具有经调节的POP多肽编码核酸表达的植物，所述植物相对于相应的野生型植物或其他对照植物具有增强的植物产量相关性状。本发明还提供了可以用于本发明方法的构建体。Furthermore, the present invention generally relates to the field of molecular biology, and relates to methods for enhancing yield-related traits of plants by modulating the expression of nucleic acids encoding POP (prolyl-oligopeptidase,Prolyl-oligopeptidase ) polypeptides in plants. The present invention also relates to plants having modulated expression of a POP polypeptide-encoding nucleic acid, which plants have enhanced plant yield-related traits relative to corresponding wild-type plants or other control plants. The invention also provides constructs that can be used in the methods of the invention.
此外，本发明一般地涉及分子生物学领域，涉及通过调节植物中编码CRL(Crampled Leaf)的核酸的表达来增强产量相关性状的方法。本发明还涉及具有经调节的CRL编码核酸表达的植物，所述植物相对于相应的野生型植物或其他对照植物具有增强的产量相关性状。本发明还提供了可以用于本发明方法的构建体。In addition, the present invention generally relates to the field of molecular biology, and relates to a method for enhancing yield-related traits by regulating the expression of nucleic acid encoding CRL (Cr amplifiedLeaf ) in plants. The present invention also relates to plants having modulated expression of a CRL-encoding nucleic acid, which plants have enhanced yield-related traits relative to corresponding wild-type plants or other control plants. The invention also provides constructs that can be used in the methods of the invention.
背景技术Background technique
不断增长的世界人口和逐渐减少的农业可用耕地推动了提高农业效率的研究之势。传统的作物和园艺学改良方法利用选育技术来鉴定具有期望特征的植物。然而，此类选育技术有若干缺陷，即这些技术一般为劳动密集型的，而且产生的植物通常含有异质的遗传组分，这些异质的遗传组分可能不总是导致期望的性状自亲本植物传递。分子生物学的进展已经使人类能够修饰动物和植物的种质。植物遗传工程需要分离和操作遗传物质(一般为DNA或RNA的形式)以及随后将遗传物质引入植物。此类技术有能力输送具有多种改良的经济、农业或园艺性状的作物或植物。A growing world population and dwindling arable land available for agriculture have fueled the research drive to improve agricultural efficiency. Traditional approaches to crop and horticultural improvement utilize selective breeding techniques to identify plants with desired characteristics. However, such selective breeding techniques have several drawbacks, namely, that these techniques are generally labor-intensive and that the resulting plants often contain a heterogeneous genetic composition that may not always result in the desired trait. The parental plant is passed on. Advances in molecular biology have enabled humans to modify the germplasm of animals and plants. Plant genetic engineering requires the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and its subsequent introduction into plants. Such technologies have the ability to deliver crops or plants with various improved economic, agricultural or horticultural traits.
具有特别经济利益的性状是增加的产量。产量通常定义为作物的可测量的具有经济价值的产出。这可以以数量和/或质量的方式进行定义。产量直接取决于若干因素，例如器官的数量和大小、植物构造(例如，分枝的数量)、种子生产、叶子衰老等等。根的发育、营养吸收、胁迫耐受性和早期活力也可以是决定产量的重要因素。因此优化上述因素可以促进作物产量的增加。A trait of particular economic interest is increased yield. Yield is usually defined as the measurable, economically valuable output of a crop. This can be defined quantitatively and/or qualitatively. Yield is directly dependent on several factors, such as number and size of organs, plant architecture (eg, number of branches), seed production, leaf senescence, and the like. Root development, nutrient uptake, stress tolerance and early vigor can also be important factors in determining yield. Therefore, optimizing the above factors can promote the increase of crop yield.
种子产量是特别重要的性状，这是因为许多植物的种子对于人类和动物营养而言至关重要。诸如玉米、稻、小麦、芸苔(canola)和大豆等作物占人类总卡路里摄取量的一半以上，或是通过对种子本身的直接消耗，或是通过对饲养自加工的种子的肉类产品的消耗。它们也可以是工业加工中所用的糖类、油类和多类代谢物的来源。种子含有胚(新的枝条和根的来源)和胚乳(萌发期和幼苗早期生长过程中胚生长的营养源)。种子的发育涉及许多基因，并且需要代谢物自根、叶和茎转移至正在生长的种子。特别是胚乳可以同化糖类、油类和蛋白质的代谢前体，将其合成为贮存高分子，以充盈籽粒。Seed yield is a particularly important trait because the seeds of many plants are essential for human and animal nutrition. Crops such as corn, rice, wheat, canola, and soybeans account for more than half of total human calorie intake, either through direct consumption of the seeds themselves or through consumption of meat products fed from processed seeds consume. They can also be a source of sugars, oils and various metabolites used in industrial processing. The seed contains the embryo (the source of new shoots and roots) and the endosperm (the nutrient source for the growth of the embryo during germination and early seedling growth). Seed development involves many genes and requires the transfer of metabolites from roots, leaves and stems to the growing seed. In particular, the endosperm can assimilate the metabolic precursors of sugars, oils and proteins, and synthesize them into storage polymers to fill the grain.
对于饲料作物如苜蓿、青贮谷物和干草，植物生物量为产量。在粮食作物中，许多产量替代参数(proxy)被使用。其中主要的是估算植物大小。根据物种以及发育阶段的不同，可以通过许多方法测量植物大小，包括植物总干重、地上干重、地上鲜重、叶面积、茎体积、植物高度、莲座(rosette)直径、叶长、根长、根质量、分蘖数和叶数。许多物种在给定的发育阶段在植物不同部分的大小之间维持保守的比例。利用这些比速增长关系可以从这些尺寸测量结果中的一个外推至另一个(如Tittonell等2005AgricEcosys&Environ 105：213)。早期发育阶段时的植物大小通常将与发育后期的植物大小有关。具有更大叶面积的较大植物通常能够比较小的植物吸收更多的光和二氧化碳，因此很可能在同期增重更多(Fasoula&Tollenaar2005Maydica 50：39)。这还不包括植物为最初实现该较大大小所已经具有的微环境或遗传优势的潜在延续。对于植物大小和生长速率，存在着强遗传组分(如ter Steege等2005Plant Physiology 139：1078)，因此对于许多不同基因型，植物在一种环境条件下的大小很可能与另一种环境条件下的大小有关(Hittalmani等2003Theoretical Applied Genetics 107：679)。由此，可以使用标准环境作为田间作物在不同地点和时间所遭遇到的多样动态环境的替代。For forage crops such as alfalfa, silage grain, and hay, plant biomass is the yield. In food crops, many yield proxy parameters are used. Chief among these is estimating plant size. Depending on the species and stage of development, plant size can be measured in a number of ways, including total plant dry weight, aboveground dry weight, aboveground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length , root mass, tiller number and leaf number. Many species maintain a conserved ratio between the sizes of different parts of the plant at a given developmental stage. Using these specific velocity relationships it is possible to extrapolate from one of these size measurements to the other (eg Tittonell et al 2005 Agric Ecosys & Environ 105:213). Plant size at early developmental stages will generally correlate with plant size at later stages of development. Larger plants with greater leaf area are generally able to absorb more light and carbon dioxide than smaller plants and are therefore likely to gain more weight over the same period (Fasoula & Tollenaar 2005 Maydica 50:39). This does not include the potential continuation of microenvironmental or genetic advantages that the plant already had to achieve this larger size in the first place. For plant size and growth rate, there is a strong genetic component (e.g. ter Steege et al. 2005 Plant Physiology 139:1078), so that for many different genotypes, the size of a plant under one environmental condition is likely to be different from that under another environmental condition. It is related to the size (Hittalmani et al. 2003 Theoretical Applied Genetics 107:679). Thus, the standard environment can be used as a substitute for the diverse dynamic environments encountered by crops in the field at different places and times.
对于许多作物而言，另一重要的性状是早期活力。提高早期活力是温带和热带稻类栽培种的现代稻类育种项目的重要目标。长根对于水栽稻的土壤锚固至关重要。在直接向涝地里播种稻米的情况下，以及在植物必须迅速透水出苗的情况下，较长的枝条均与活力有关。在进行条播的情况下，较长的中胚轴和胚芽鞘对于优良的出苗至关重要。改造植物早期活力的能力在农业上将具有极其重要的意义。例如，一直以来早期活力弱限制了在欧洲大西洋地区引入基于玉米带种质的玉米(玉蜀黍，Zea mays L.)杂交种。Another important trait for many crops is early vigor. Improving early vigor is an important goal of modern rice breeding programs of temperate and tropical rice cultivars. Long roots are essential for soil anchorage in hydroponics. Longer shoots were associated with vigor both when rice was sown directly into flooded fields, and when plants had to emerge quickly through water. In case of drill seeding, longer mesocotyls and coleoptiles are essential for good emergence. The ability to engineer early plant vigor would be of enormous importance in agriculture. For example, poor early vigor has historically limited the introduction of maize (Zea mays L.) hybrids based on Corn Belt germplasm in the European Atlantic region.
收获指数为种子产量与地上干重的比值，其在许多环境条件下相对稳定，因此在植物大小和粮食产量之间通常能够获得比较稳固的相关性(例如Rebetzke等2002Crop Science 42：739)。这些程序固有地联系在一起，因为大多数粮食生物量取决于植物叶和茎当前或贮存的光合作用生产力(Gardener等(1985)Physiology of Crop Plants.Iowa State UniversityPress，68-73页)。因此，对植物大小的选择，甚至是在发育早期阶段的选择，已经用作为未来潜在产量的指标(如Tittonell等2005Agric Ecosys&Environ 105：213)。当测试遗传差异对胁迫耐受性的影响时，温室或植物培养室环境与田地相比具有固有的优势：即能够使土壤性能、温度、水和养分可利用率以及光强度标准化的能力。不过，因不良授粉(由缺乏风力或昆虫导致)或因空间不足以让成熟根或冠层生长等等而对产量造成的人为局限性，会限制这些受控环境在测试产量差异中的应用。因此，在培养室或温室中在标准条件下测量早期发育阶段的植物大小，是指示潜在遗传产量优势的标准方法。Harvest index, the ratio of seed yield to aboveground dry weight, is relatively stable under many environmental conditions, so a relatively robust correlation can usually be obtained between plant size and grain yield (eg Rebetzke et al. 2002 Crop Science 42:739). These procedures are inherently linked because most food biomass depends on the current or stored photosynthetic productivity of plant leaves and stems (Gardener et al. (1985) Physiology of Crop Plants. Iowa State University Press, pp. 68-73). Thus, selection on plant size, even at early stages of development, has been used as an indicator of future yield potential (eg Tittonell et al. 2005 Agric Ecosys & Environ 105:213). When testing the effects of genetic differences on stress tolerance, the greenhouse or plant chamber environment has inherent advantages over the field: namely, the ability to normalize soil properties, temperature, water and nutrient availability, and light intensity. However, artificial limitations on yield due to poor pollination (caused by lack of wind or insects) or insufficient space for mature root or canopy growth, etc., limit the use of these controlled environments to test yield differences. Therefore, measuring plant size at early developmental stages under standard conditions in a culture chamber or greenhouse is a standard method for indicating potential genetic yield advantages.
再一特别重要的性状为提高的非生物胁迫耐受性。非生物胁迫是全世界作物损失的主要原因，使大多数主要作物植物平均产量降低50％以上(Wang等，Planta(2003)218：1-14)。非生物胁迫可以因为干旱、盐度、极端温度、化学毒性、营养物(大量元素和/或微量元素)的过量或缺乏、辐射及氧化胁迫引起。增强非生物胁迫植物耐受性的能力将对全世界农场主带来重大的经济利益，并将使人们能够在否则将不可能进行作物栽培的地区和不利条件下进行作物栽培。A further particularly important trait is increased tolerance to abiotic stresses. Abiotic stress is a major cause of crop loss worldwide, reducing the average yield of most major crop plants by more than 50% (Wang et al., Planta (2003) 218: 1-14). Abiotic stresses can be caused by drought, salinity, temperature extremes, chemical toxicity, excess or deficiency of nutrients (macroelements and/or microelements), radiation and oxidative stress. The ability to increase plant tolerance to abiotic stress would be of major economic benefit to farmers worldwide and would enable the cultivation of crops in areas and under adverse conditions that would otherwise not be possible.
因此通过优化上述因素之一可以增加作物产量。Crop yields can therefore be increased by optimizing one of the aforementioned factors.
视最终用途而定，对某些产量性状的修饰可能优于对其他产量性状的修饰。例如，对于诸如饲料或木材生产或者生物燃料源等应用，可能期望植物营养部分的增加，而对于诸如面粉、淀粉或油料生产等应用，可能特别期望种子参数的增强。即便是在种子参数之中，取决于应用，一些参数也可能优于其它参数。多种机制可以促成增加的种子产量，无论是以增加的种子大小还是以增加的种子数量的形式。Depending on the end use, modifications to some yield traits may be preferable to modifications to other yield traits. For example, for applications such as feed or wood production, or biofuel sources, an increase in the vegetative fraction of the plant may be desired, while for applications such as flour, starch or oilseed production, enhancement of seed parameters may particularly be desired. Even among the seed parameters, some parameters may be better than others depending on the application. Various mechanisms may contribute to increased seed yield, whether in the form of increased seed size or increased seed number.
增加植物产量和/或产量相关性状(种子产量和/或生物量)的一种方法可以是修饰植物的内在生长机制，如细胞周期或者参与植物生长或防御机制的多种信号传递路径。One approach to increasing plant yield and/or yield-related traits (seed yield and/or biomass) may be to modify intrinsic growth mechanisms of plants, such as the cell cycle or various signaling pathways involved in plant growth or defense mechanisms.
现已发现，可通过调节alfin-样多肽编码核酸在植物中的表达来增强植物对多种非生物胁迫的耐受性。It has now been found that the tolerance of plants to various abiotic stresses can be enhanced by regulating the expression of alfin-like polypeptide-encoding nucleic acids in plants.
现已发现，可通过调节YRP多肽编码核酸在植物中的表达来增强植物对多种非生物胁迫的耐受性。It has now been found that the tolerance of plants to various abiotic stresses can be enhanced by regulating the expression of nucleic acids encoding YRP polypeptides in plants.
现已发现，可通过增加编码Brevis Radix样(BRXL)多肽的核酸序列在植物中的表达，相对于对照植物而增强植物的多种产量相关性状。增强的产量相关性状包括如下一种或多种：增加的植株高度和增加的千粒重(TKW)。It has now been found that by increasing expression in a plant of a nucleic acid sequence encoding aBrevisRadix- like (BRXL) polypeptide, various yield-related traits can be enhanced in plants relative to control plants. Enhanced yield-related traits include one or more of: increased plant height and increased thousand kernel weight (TKW).
现已发现，可通过调节silky-1样多肽编码核酸在植物中的表达来增强植物对多种非生物胁迫的耐受性。It has now been found that the tolerance of plants to various abiotic stresses can be enhanced by regulating the expression of nucleic acids encoding silky-1-like polypeptides in plants.
现已发现，可通过在植物中调节ARP6多肽编码核酸在植物中的表达来改善植物的多种生长特征。It has now been found that various growth characteristics of plants can be improved by modulating expression in plants of nucleic acids encoding ARP6 polypeptides in plants.
现已发现，可通过在植物中调节POP编码核酸在植物中的表达来改善植物的多种生长特征。It has now been found that various growth characteristics of plants can be improved by modulating expression in plants of POP-encoding nucleic acids in plants.
现已发现，可通过在植物中调节CRL(Crumpled Leaf)编码核酸在植物中的表达来改善植物的多种产量相关性状。It has been found that various yield-related traits of plants can be improved by regulating the expression ofnucleic acid encoding CRL (CrumpledLeaf ) in plants.
背景background
1.Alfin样多肽1. Alfin-like peptide
在从植物到动物的许多调节蛋白中发现PHD指，Cys4-His-Cys3锌指，其常与染色质介导的转录调控相关。已证实PHD指在酵母、植物和动物细胞中激活转录(Halbach等人，Nucleic Acids Res.2000 September 15；28(18)：3542-3550)。The PHD finger, the Cys4 -His-Cys3 zinc finger, is found in many regulatory proteins from plants to animals, often associated with chromatin-mediated transcriptional regulation. PHD fingers have been shown to activate transcription in yeast, plant and animal cells (Halbach et al., Nucleic Acids Res. 2000 September 15;28(18):3542-3550).
Alfin样来源的锌指结构域属于PHD指结构域家族(R.Aasland，等，Trends Biochem Sci(1995)20：56-9)。据推测，Alfin样PHD结构域起着以EDTA敏感的方式结合DNA的作用，从而推断，对于在GNGGTG或GTGGNG的核心六聚体基序上的结合，是需要锌的(D.Bastola，等，PlantMol Biol.(1998)38：1123-35)。在拟南芥中鉴定了8个Alfin-样因子(ALF)基因(J.L.Riechmann，等，Science(2000)290：2105-10)。表达alfin样的反义形式导致了转基因苜蓿生长更弱，而通过组成型启动子进行的组成型过表达在正常和盐胁迫条件下均增强了根生长(I.Winicov Planta(2000)210：416-22)。Zinc finger domains of Alfin-like origin belong to the PHD finger domain family (R. Aasland, et al., Trends Biochem Sci (1995) 20:56-9). It is speculated that the Alfin-like PHD domain functions to bind DNA in an EDTA-sensitive manner, leading to the conclusion that zinc is required for binding at the core hexamer motifs of GNGGTG or GTGGNG (D. Bastola, et al. Plant Mol Biol. (1998) 38: 1123-35). EightAlfin -like factor (ALF) genes were identified in Arabidopsis (JLRiechmann, et al., Science (2000) 290:2105-10). Expression of an alfin-like antisense form resulted in weaker growth of transgenic alfalfa, while constitutive overexpression through a constitutive promoter enhanced root growth under both normal and salt stress conditions (I. Winicov Planta (2000) 210:416 -twenty two).
2.YRP多肽2. YRP polypeptide
大麦(Hordeum vulgare)的YRP(SEQ ID NO：11和13)是编码GARP类转录因子成员的转录因子。YRP (SEQ ID NO: 11 and 13) of barley (Hordeum vulgare) is a transcription factor encoding a member of the GARP class of transcription factors.
3.Brevis Radix样(BRXL)多肽3. Brevis Radix-like (BRXL) polypeptide
Brevis Radix(BRX)多肽通过拟南芥登录号Umkirch-1(Uk-1)的天然功能丧失性等位基因而得到鉴定，该多肽因扰乱植物激素油菜素类固醇和生长素信号转导途径而导致减小的根分生组织大小、减小的成熟细胞大小，以及由此减小的根生长(Mouchel等(2004)Genes Dev 18：700-714)。The B revisRadix (BRX ) polypeptide, identified by the natural loss-of-function allele of Arabidopsis accession number Umkirch-1 (Uk-1), is responsible for disrupting the plant hormone brassinosteroid and auxin signaling pathway leading to reduced root meristem size, reduced mature cell size, and thus reduced root growth (Mouchel et al. (2004) Genes Dev 18:700-714).
BRX和旁系同源物BRX样(BRXL)属于一个保守的植物特异性基因家族(统称为BRXL)，编码预测直接或间接调节转录的蛋白质。BRXL基因存在于可获得数据的所有高等植物中，但在单细胞生物和动物中不存在。在拟南芥、白杨(Populus trichocarpa)和稻(Oryza sativa)的完全测序的植物基因组中，可发现5个BRXL基因(Briggs等(2006)Plant Physiol 140：1307-1316)。BRX and the paralog BRX-like (BRXL) belong to a conserved family of plant-specific genes (collectively referred to as BRXL) encoding proteins predicted to directly or indirectly regulate transcription. The BRXL gene is present in all higher plants for which data are available, but absent in unicellular organisms and animals. In the fully sequenced plant genomes of Arabidopsis, Populus trichocarpa and Oryza sativa, five BRXL genes can be found (Briggs et al. (2006) Plant Physiol 140: 1307-1316).
在BRX家族蛋白中可区别出高度保守的4个结构域。在物种内和物种之间BRX家族蛋白质之间的同源性在这些区域中是特别保守的：Four highly conserved domains can be distinguished among BRX family proteins. Homology between BRX family proteins within and between species is particularly conserved in these regions:
1.在N端上，分别约10和25个氨基酸的两个短结构域是保守的，包含保守的Cys，其间距(spacing)指示潜在的锌结合基序。1. On the N-terminus, two short domains of about 10 and 25 amino acids respectively are conserved, containing conserved Cys, the spacing of which indicates potential zinc-binding motifs.
2.BRX家族蛋白的中间区域包含约65个氨基酸的高度保守结构域。2. The middle region of BRX family proteins contains a highly conserved domain of about 65 amino acids.
3.存在约60个氨基酸的第二高度保守结构域，其与第一中间结构域同源，构成新型串联重复，这是BRX家族蛋白的主要特征(从而称为BRX结构域)。3. There is a second highly conserved domain of about 60 amino acids, which is homologous to the first intermediate domain, constituting a novel tandem repeat, which is the main feature of BRX family proteins (thereby called BRX domain).
已在保守的BRX结构域中鉴定了DNA结合和蛋白质-蛋白质相互作用结构域所特征性的α螺旋区。酵母双杂交实验显示，BRX结构域是新型蛋白质-蛋白质相互作用结构域，其可能介导BRXL以及还有PRAF样(PH、RCC1和FYVE)蛋白家族的内部和/或之间的同二聚化和异二聚化(Briggs等(2006)Plant Physiol 140：1307-1316；van Leeuwen等(2004)TrendsPlant Sci 9：378-384)。PRAF样蛋白也包含染色体浓缩1(RCC1)重复序列的调节因子，其通常提供鸟嘌呤核苷酸交换活性。An alpha-helical region characteristic of DNA-binding and protein-protein interaction domains has been identified in the conserved BRX domain. Yeast two-hybrid experiments revealed that the BRX domain is a novel protein-protein interaction domain that may mediate homogeneity within and/or between BRXL and also PRAF-like (PH ,RCC1 , andFYVE ) protein families. Dimerization and heterodimerization (Briggs et al. (2006) Plant Physiol 140:1307-1316; van Leeuwen et al. (2004) Trends Plant Sci 9:378-384). PRAF-like proteins also contain regulators of the chromosomal condensation 1 (RCC1) repeat, which normally provides guanine nucleotide exchange activity.
在美国专利7214786“Nucleic acid molecules and other moleculesassociated with plants and uses thereof for plant improvement”中，描述了编码BRXL多肽的核酸序列(SEQ IDs NO：35674、30290、17003)和包含此类核酸的构建体。公开的重组多核苷酸和重组多肽用于产生转基因植物，以产生具有改善的性质的植物。在美国专利7365185“Genomic plantsequences and uses thereof”中，描述了BRXL多肽的稻核酸启动子序列(SEQ IDs NO：59178、70484、70442、37078、78410、64873)和包含此类启动子的构建体。该发明还公开了包含稻基因组启动子序列的组合物、转化的宿主细胞、转基因植物和种子、以及用于制备和使用它们的方法。In U.S. Patent 7214786 "Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement", a nucleic acid sequence (SEQ IDs NO: 35674, 30290, 17003) encoding a BRXL polypeptide and a construct comprising such a nucleic acid are described. The disclosed recombinant polynucleotides and recombinant polypeptides are used to generate transgenic plants to produce plants with improved properties. In US Patent 7365185 "Genomic plants sequences and uses thereof", the rice nucleic acid promoter sequences (SEQ IDs NO: 59178, 70484, 70442, 37078, 78410, 64873) of BRXL polypeptides and constructs comprising such promoters are described. The invention also discloses compositions comprising rice genome promoter sequences, transformed host cells, transgenic plants and seeds, and methods for making and using them.
4.silky-1样多肽4. silky-1-like polypeptide
Silky-1是MADS转录因子家族的成员，其参与花发育。Silky-1 is a member of the MADS family of transcription factors involved in flower development.
5.ARP6多肽5. ARP6 polypeptide
肌动蛋白相关蛋白(ARP)构成一个真核蛋白质家族，其一级序列显示与常规肌动蛋白同源。肌动蛋白具有良好表征的细胞骨架作用，而ARP参与细胞质和细胞核中的多种细胞功能。例如已知细胞质ARP在许多真核细胞中参与分枝的肌动蛋白丝的装配和动力蛋白介导的小泡的运动。细胞核ARP是参与转录调控的多种染色质修饰复合物的成分。在植物中，例如最近已描述了SWR1/SRCAP样复合物的存在。此类复合物似乎使核小体内的蛋白质间和蛋白质-DNA间的相互作用去稳定化，从而允许染色体重建，由此影响基因表达。酵母和哺乳动物ARP6蛋白分别与SWR1和SRCAP复合物起作用，其使组蛋白变体H2A.Z沉积入染色质。拟南芥ARP6与ARP6、PIE1和SEF蛋白相互作用，表明ARP6功能在植物界中也是保守的(March-Diaz，R.等2007.Plant Physiol.143，893-901)。AtARP6的敲除突变导致拟南芥中的许多基因表达的误调控和早期开花及矮化表型(Deal 2007，The Plant Cell，第19卷：74-83)。Actin-related proteins (ARPs) constitute a family of eukaryotic proteins whose primary sequence shows homology to conventional actin. Actin has a well-characterized cytoskeletal role, whereas ARP is involved in a variety of cellular functions in the cytoplasm and nucleus. For example, cytoplasmic ARP is known to be involved in the assembly of branched actin filaments and dynein-mediated vesicle movement in many eukaryotic cells. Nuclear ARPs are components of multiple chromatin-modifying complexes involved in transcriptional regulation. In plants, for example, the presence of a SWR1/SRCAP-like complex has recently been described. Such complexes appear to destabilize protein-protein and protein-DNA interactions within the nucleosome, allowing chromosomal remodeling and thereby affecting gene expression. Yeast and mammalian ARP6 proteins act in complex with SWR1 and SRCAP, respectively, which deposit the histone variant H2A.Z into chromatin. Arabidopsis ARP6 interacts with ARP6, PIE1 and SEF proteins, suggesting that ARP6 function is also conserved in the plant kingdom (March-Diaz, R. et al. 2007. Plant Physiol. 143, 893-901). Knockout mutations of AtARP6 lead to misregulation of expression of many genes and early flowering and dwarf phenotypes in Arabidopsis (Deal 2007, The Plant Cell, Vol. 19: 74-83).
ARP和肌动蛋白具有共同的三级结构，以称为肌动蛋白折叠的核苷酸结合口袋为中心(Kabach等1995.FASEB J.9，167-1745)。ARP被分成在广泛的真核生物中高度保守的若干种类或亚家族。每一个种类或亚家族的区别特征在于其与常规肌动蛋白的相似性程度(Kandasamy等2004Trends Plant Sci 9：196-202)。ARP and actin share a common tertiary structure centered on a nucleotide-binding pocket called the actin fold (Kabach et al. 1995. FASEB J. 9, 167-1745). ARPs are divided into several classes or subfamilies that are highly conserved across a wide range of eukaryotes. Each species or subfamily is distinguished by its degree of similarity to conventional actin (Kandasamy et al. 2004 Trends Plant Sci 9: 196-202).
6.POP多肽6. POP polypeptide
蛋白酶在多肽链的末端(外肽酶)或在多肽链内(内肽酶)催化肽键的水解。根据它们的结构保守性，可以分类它们，参见例如MEROPS数据库(Rawlings等，Nucleic Acids Research 34，D270-272，2006)。丝氨酸蛋白酶在其活性部位具有在底物的水解中起作用的丝氨酸，并且在植物中形成了最大一组的蛋白酶。在丝氨酸蛋白酶组内，可区分若干亚组，例如枯草杆菌蛋白酶家族、胰凝乳蛋白酶家族、D-Ala-D-Ala羧肽酶B家族或脯氨酰寡肽酶家族。Proteases catalyze the hydrolysis of peptide bonds either at the ends of the polypeptide chain (exo-peptidases) or within the polypeptide chain (endopeptidases). They can be classified according to their structural conservation, see for example the MEROPS database (Rawlings et al., Nucleic Acids Research 34, D270-272, 2006). Serine proteases have in their active site a serine that plays a role in the hydrolysis of substrates and form the largest group of proteases in plants. Within the group of serine proteases, several subgroups can be distinguished, such as the subtilisin family, the chymotrypsin family, the D-Ala-D-Ala carboxypeptidase B family or the prolyl oligopeptidase family.
脯氨酰-寡肽酶据推定在生物活性肽的形成、加工和降解中起作用，并且已在细菌、古细菌以及在真核生物中进行了描述。在脯氨酰-寡肽酶组中，可区分4个亚家族：脯氨酰寡肽酶(S9A)、二肽基肽酶IV(S9B)、氨酰基肽酶(S9C)和谷氨酰基内肽酶(S9D)。Prolyl-oligopeptidases are putatively involved in the formation, processing and degradation of biologically active peptides and have been described in bacteria, archaea and in eukaryotes. Within the group of prolyl-oligopeptidases, four subfamilies can be distinguished: prolyl oligopeptidases (S9A), dipeptidyl peptidases IV (S9B), aminoacyl peptidases (S9C) and glutamyl endopeptidases Peptidase (S9D).
尽管已鉴定了如此多的蛋白酶，然而关于这些酶的底物知之甚少。因此，蛋白酶的功能和调节也几乎未被表征。Despite so many proteases being identified, very little is known about the substrates of these enzymes. Consequently, the function and regulation of proteases are also largely uncharacterized.
7.Crumpled Leaf(CRL)多肽7. Crumpled Leaf (CRL) polypeptide
Crumpled leaf(crl)是给予一种拟南芥突变体的名称，所述突变体中所有植物器官和质体的分裂具有异常的形态发生。组织学分析揭示在具有crl突变的植物的茎端分生组织(SAM)、根尖和胚中细胞分裂面是扭曲的。此外，在crl突变体中，花序轴的皮层和内皮层细胞和根内皮层细胞的分化模式被扰乱。这些结果提示，在crl突变体中观察到的形态学异常是由异常细胞分裂和分化造成的。此外，crl突变体的细胞包含减少数量的扩大的质体，这表明质体的分裂在crl中被抑制。负责该表型的突变的基因称为CRL(Crumpled leaf)。CRL基因编码具有30kDa的分子量的蛋白质，其位于质体被膜中。CRL蛋白在多种植物物种包括蕨类植物中以及在蓝细菌(cyanobacteria)中是保守的。拟南芥的CRL蛋白具有位于氨基酸19至36之间的推定膜结构域和氨基酸残基42至236之间的保守结构域。该结构域在其他植物物种中存在的CRL蛋白之间是高度保守的。Crumpled leaf (crl) is the name given to an Arabidopsis mutant in which division of all plant organs and plastids has abnormal morphogenesis. Histological analysis revealed distorted cell division planes in the shoot apical meristem (SAM), root tip and embryo of plants with the crl mutation. Furthermore, the differentiation pattern of cortical and endodermal cells of the inflorescence axis and root endothelial cells was disrupted in crl mutants. These results suggest that the morphological abnormalities observed in crl mutants result from abnormal cell division and differentiation. Furthermore, cells of crl mutants contained reduced numbers of enlarged plastids, suggesting that division of plastids is repressed in crl. The mutated gene responsible for this phenotype is called CRL (Crumpled leaf). The CRL gene encodes a protein with a molecular weight of 30 kDa, which is located in the plastid envelope. CRL proteins are conserved in various plant species including ferns and in cyanobacteria. The CRL protein of Arabidopsis has a putative membrane domain located between amino acids 19 to 36 and a conserved domain between amino acid residues 42 to 236. This domain is highly conserved among CRL proteins present in other plant species.
发明概述Summary of the invention
1.Alfin样多肽1. Alfin-like peptide
令人惊讶地，现已发现，调节编码alfin样多肽的核酸的表达可以产生相对于对照植物具有增强的对多种非生物胁迫的耐受性的植物。Surprisingly, it has now been found that modulating the expression of a nucleic acid encoding an alfin-like polypeptide can give plants having increased tolerance to various abiotic stresses relative to control plants.
根据一个实施方案，本发明提供用于相对于对照植物的耐受性而增强植物对多种非生物胁迫的耐受性的方法，包括调节编码alfin样多肽的核酸在植物中的表达。According to one embodiment, the present invention provides a method for increasing the tolerance of a plant to various abiotic stresses relative to the tolerance of control plants, comprising modulating expression in a plant of a nucleic acid encoding an alfin-like polypeptide.
2.YRP多肽2. YRP polypeptide
令人惊讶地，现已发现，调节编码YRP多肽的核酸的表达可以产生相对于对照植物具有增强的对多种非生物胁迫的耐受性的植物。Surprisingly, it has now been found that modulating the expression of a nucleic acid encoding a YRP polypeptide can give plants having increased tolerance to various abiotic stresses relative to control plants.
根据一个实施方案，本发明提供用于相对于对照植物的耐受性而增强植物对多种非生物胁迫的耐受性的方法，包括调节编码YRP多肽的核酸在植物中的表达。According to one embodiment, the present invention provides a method for enhancing the tolerance of a plant to various abiotic stresses relative to the tolerance of control plants, comprising modulating expression in a plant of a nucleic acid encoding a YRP polypeptide.
3.Brevis Radix样(BRXL)多肽3. Brevis Radix-like (BRXL) polypeptide
令人惊讶地，现已发现，增加编码如本文中定义的BRXL多肽的核酸序列在植物中的表达可以产生相对于对照植物具有增强的产量相关性状的植物。Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid sequence encoding a BRXL polypeptide as defined herein gives plants having enhanced yield-related traits relative to control plants.
根据一个实施方案，提供用于相对于对照植物而增强植物的产量相关性状的方法，包括增加编码如本文中定义的BRXL多肽的核酸序列在植物中的表达。所述增强的产量相关性状包括如下一个或多个：增加的植物高度和增加的千粒重(TKW)。According to one embodiment, there is provided a method for enhancing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a BRXL polypeptide as defined herein. Said enhanced yield-related traits include one or more of: increased plant height and increased thousand-kernel weight (TKW).
4.silky-1样多肽4. silky-1-like polypeptide
令人惊讶地，现已发现，调节编码silky-1样多肽的核酸的表达可以产生相对于对照植物具有增强的对多种非生物胁迫的耐受性的植物。Surprisingly, it has now been found that modulating the expression of a nucleic acid encoding a silky-1-like polypeptide gives plants having increased tolerance to various abiotic stresses relative to control plants.
根据一个实施方案，提供用于相对于对照植物的耐受性而增强植物对多种非生物胁迫的耐受性的方法，包括调节编码silky-1样多肽的核酸在植物中的表达。According to one embodiment, there is provided a method for increasing the tolerance of a plant to various abiotic stresses relative to the tolerance of control plants, comprising modulating expression in a plant of a nucleic acid encoding a silky-1-like polypeptide.
5.ARP6多肽5. ARP6 polypeptide
令人惊讶地，现已发现，调节编码ARP6多肽的核酸的表达可以产生相对于对照植物具有增强的产量相关性状的植物。Surprisingly, it has now been found that modulating the expression of a nucleic acid encoding an ARP6 polypeptide gives plants having enhanced yield-related traits relative to control plants.
根据一个实施方案，提供用于相对于对照植物而增强植物的产量相关性状的方法，包括调节编码ARP6多肽的核酸在植物中的表达。According to one embodiment, there is provided a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding an ARP6 polypeptide.
6.POP多肽6. POP polypeptide
令人惊讶地，现已发现，调节编码POP多肽的核酸的表达可以产生相对于对照植物具有增强的产量相关性状的植物。Surprisingly, it has now been found that modulating the expression of a nucleic acid encoding a POP polypeptide gives plants having enhanced yield-related traits relative to control plants.
根据一个实施方案，提供用于相对于对照植物而增强植物的产量相关性状的方法，包括调节编码POP多肽的核酸在植物中的表达。According to one embodiment, there is provided a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a POP polypeptide.
7.Crumpled Leaf(CRL)多肽7. Crumpled Leaf (CRL) polypeptide
令人惊讶地，现已发现，调节编码CRL多肽的核酸的表达可以产生相对于对照植物具有增强的产量相关性状的植物。Surprisingly, it has now been found that modulating the expression of a nucleic acid encoding a CRL polypeptide gives plants having enhanced yield-related traits relative to control plants.
根据一个实施方案，提供用于相对于对照植物而增强植物的产量相关性状的方法，包括调节编码CRL多肽的核酸在植物中的表达。According to one embodiment, there is provided a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a CRL polypeptide.
定义definition
多肽/蛋白质Peptide/Protein
术语“多肽”和“蛋白质”在文中可互换使用，是指通过肽键连接起来的、任意长度的氨基酸的聚合物。The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length linked by peptide bonds.
多核苷酸/核酸/核酸序列/核苷酸序列polynucleotide/nucleic acid/nucleic acid sequence/nucleotide sequence
术语“多核苷酸”、“核酸序列”、“核苷酸序列”、“核酸”、“核酸分子”在文中可互换使用，是指任何长度的无支链形式的核苷酸聚合物，所述核苷酸可以为核糖核苷酸或脱氧核糖核苷酸或者两者的组合。The terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence", "nucleic acid", "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleotides of any length in unbranched form, The nucleotides may be ribonucleotides or deoxyribonucleotides or a combination of both.
对照植物control plants
选择适宜的对照植物是实验设置的常规部分，并且可以包括相应的野生型植物或不合目的基因的相应植物。对照植物一般与待评估植物为相同的植物物种，或者甚至为同一品种。对照植物还可以是待评估植物的无效合子。无效合子是因分离而失去转基因的个体。如本文所用的“对照植物”不仅指完整植物，而且还指植物部分，包括种子和种子部分。Selection of suitable control plants is a routine part of the experimental setup and may include corresponding wild-type plants or corresponding plants not suitable for the gene of interest. The control plants are generally the same plant species, or even the same variety, as the plants to be evaluated. Control plants can also be nullzygotes of the plants to be evaluated. Nullizygotes are individuals who have lost the transgene by segregation. A "control plant" as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
同源物homologue
蛋白质的“同源物”包括肽、寡肽、多肽、蛋白质和酶，其相对于所讨论的未修饰蛋白质具有氨基酸取代、缺失和/或插入，并且与其源自的未修饰蛋白质具有相似的生物活性和功能活性。"Homologues" of proteins include peptides, oligopeptides, polypeptides, proteins and enzymes which have amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and which have a similar biological activity and functional activity.
缺失是指从蛋白质中除去一个或多个氨基酸。Deletion refers to the removal of one or more amino acids from a protein.
插入是指在蛋白质的预定位置引入一个或多个氨基酸残基。插入可以包括N-末端和/或C-末端融合，以及单个或多个氨基酸的序列内插入。一般，氨基酸序列内的插入将小于N-或C-末端的融合，约1到10个残基左右。N-或C-末端融合蛋白或肽的实例包括在酵母双杂交系统中应用的转录激活因子的结合结构域或激活结构域、噬菌体外壳蛋白、(组氨酸)-6-标签、谷胱甘肽S-转移酶标签、蛋白质A、麦芽糖结合蛋白、二氢叶酸还原酶、Tag·100表位、c-myc表位、
取代是指蛋白质中的氨基酸用具有相似特性(如相似的疏水性、亲水性、抗原性、形成或打破α螺旋结构或β片层结构的倾向)的其他氨基酸替换。氨基酸取代一般是单残基的取代，但是视施加于多肽上的功能性限制而定也可以是成簇取代；插入通常在大约1到10个氨基酸残基的数量级。氨基酸取代优选为保守氨基酸取代。保守取代表在本领域公知(参见例如Creighton(1984)Proteins.W.H.Freeman and Company(编辑)和下表1)。Substitution refers to the replacement of amino acids in proteins with other amino acids that have similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or break alpha helices or beta sheets). Amino acid substitutions are typically of single residues, but may also be clustered depending on functional constraints imposed on the polypeptide; insertions are usually on the order of about 1 to 10 amino acid residues. Amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see, eg, Creighton (1984) Proteins. W.H. Freeman and Company (eds.) and Table 1 below).
表1：保守氨基酸取代的实例Table 1: Examples of Conservative Amino Acid Substitutions
可通过本领域公知的肽合成技术，如固相肽合成法等，或通过重组DNA操作，容易地进行氨基酸取代、缺失和/或插入。用于产生蛋白质的取代、插入或缺失变体的DNA序列操作方法在本领域公知。例如，本领域的技术人员公知在DNA预定位置进行取代突变的技术，包括M13诱变、T7-Gen体外诱变(USB，Cleveland，OH)、QuickChange定点诱变(Stratagene，San Diego，CA)、PCR介导的定点诱变或其他定点诱变方案。Amino acid substitutions, deletions and/or insertions can be readily performed by peptide synthesis techniques known in the art, such as solid phase peptide synthesis, etc., or by recombinant DNA manipulation. Methods of DNA sequence manipulation for generating substitution, insertion or deletion variants of proteins are well known in the art. For example, techniques for performing substitution mutations at predetermined positions in DNA are known to those skilled in the art, including M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange site-directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
衍生物derivative
“衍生物”包括肽、寡肽、多肽，与蛋白质如目的蛋白质的天然形式的氨基酸序列相比，其可以包括用非天然氨基酸残基进行的氨基酸取代、或者添加非天然氨基酸残基。蛋白质的“衍生物”还包括肽、寡肽、多肽，与多肽的天然形式的氨基酸序列相比，其可以包括天然改变的(糖基化、酰基化、异戊烯化、磷酸化、肉豆蔻酰化、硫酸化等)或非天然改变的氨基酸残基。衍生物与其源自的氨基酸序列相比，还可以包括一个或多个非氨基酸取代或添加，例如共价或非共价地结合于氨基酸序列的报告分子或其他配体，例如与氨基酸序列结合以有利于其检测的报告分子，以及相对于天然蛋白质的氨基酸序列而言非天然的氨基酸残基。此外，“衍生物”还可以包括天然形式的蛋白质与标签肽(tagging peptide)例如FLAG、HIS6或硫氧还蛋白的融合物(关于标签肽的综述，参见Terpe，Appl.Microbiol.Biotechnol.60，523-533，2003)。"Derivatives" include peptides, oligopeptides, polypeptides, which may include amino acid substitutions with unnatural amino acid residues, or addition of unnatural amino acid residues, compared to the amino acid sequence of a protein, such as a protein of interest, in its native form. "Derivatives" of proteins also include peptides, oligopeptides, polypeptides, which may include naturally altered (glycosylation, acylation, prenylation, phosphorylation, myristic acylation, sulfation, etc.) or unnaturally altered amino acid residues. A derivative may also comprise one or more non-amino acid substitutions or additions compared to the amino acid sequence from which it is derived, for example a reporter or other ligand covalently or non-covalently bound to the amino acid sequence, for example bound to the amino acid sequence to reporter molecules to facilitate its detection, and amino acid residues that are unnatural relative to the amino acid sequence of the native protein. Furthermore, "derivatives" may also include fusions of the native form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).
直向同源物/旁系同源物Orthologues/Paralogues
直向同源物和旁系同源物涵盖用于描述基因的祖先关系的进化概念。旁系同源物为相同物种内的基因，其起源自祖先基因的复制；而直向同源物为来自不同生物体的基因，其通过物种形成起源，并且也源自于共同的祖先基因。Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral relationship of genes. Paralogs are genes within the same species that arose from the duplication of an ancestral gene, while orthologues are genes from different organisms that originated by speciation and also arose from a common ancestral gene.
结构域domain
术语“结构域”是指在进化相关蛋白质的序列比对中，在特定位置上保守的一组氨基酸。尽管其他位置上的氨基酸可能因同源物不同而改变，但是在特定位置上高度保守的氨基酸则意味着对于蛋白质结构、稳定性或功能而言很可能是必不可少的氨基酸。“结构域”通过在蛋白质同源物家族的比对序列中其高度的保守性而鉴定，其能够用作为标识符以确定任何所讨论的多肽是否属于先前鉴定到的多肽家族。The term "domain" refers to a group of amino acids conserved at a specific position in an alignment of evolutionarily related proteins. While amino acids at other positions may vary with homologues, amino acids that are highly conserved at a particular position mean that they are likely to be essential for protein structure, stability, or function. A "domain" is identified by its high degree of conservation in aligned sequences of a family of protein homologues, which can be used as an identifier to determine whether any polypeptide in question belongs to a previously identified polypeptide family.
基序/共有序列/标签序列(signature)Motif/Consensus/Tag sequence (signature)
术语“基序”或“共有序列”或“标签序列”是指进化相关蛋白质序列中短的保守区域。基序常常是结构域的高度保守的部分，但也可以包括仅仅部分的结构域，或者可以是位于保守结构域之外(若基序的所有氨基酸都落在所定义的结构域之外的话)。The terms "motif" or "consensus sequence" or "signature sequence" refer to short conserved regions in evolutionarily related protein sequences. Motifs are often highly conserved parts of domains, but may also include only part of a domain, or may be located outside a conserved domain (if all amino acids of the motif fall outside a defined domain) .
杂交hybridize
本文定义的术语“杂交”指其中基本同源互补的核苷酸序列彼此退火的过程。杂交过程能够完全在溶液中发生，即互补的核酸都处在溶液中。杂交过程也能够这样进行，即互补核酸之一固定于基质，如磁珠、琼脂糖珠或任何其它树脂上。此外，杂交过程也能够这样进行，即其中互补核酸之一固定在固相支持物如硝酸纤维素或尼龙膜上，或者通过例如照相平板印刷术固定在例如硅质玻璃支持物上(后者称为核酸阵列或微阵列，或称为核酸芯片)。为了使杂交发生，通常使核酸分子热变性或化学变性，以使双链解链成两条单链，和/或除去单链核酸中的发夹结构或其它二级结构。The term "hybridization" as defined herein refers to a process in which substantially homologous and complementary nucleotide sequences anneal to each other. The hybridization process can occur entirely in solution, ie the complementary nucleic acids are both in solution. The hybridization process can also be performed with one of the complementary nucleic acids immobilized on a matrix such as magnetic beads, sepharose beads or any other resin. In addition, the hybridization process can also be carried out in which one of the complementary nucleic acids is immobilized on a solid support such as nitrocellulose or nylon membrane, or on a support such as silica glass by e.g. photolithography (the latter is called Nucleic acid array or microarray, or nucleic acid chip). In order for hybridization to occur, nucleic acid molecules are typically thermally or chemically denatured to melt the double strand into two single strands and/or to remove hairpins or other secondary structures in single-stranded nucleic acids.
术语“严格性”是指进行杂交的条件。杂交的严格性受诸如温度、盐浓度、离子强度和杂交缓冲液组成等条件的影响。通常，在确定的离子强度和pH，对于特定序列而言，低严格条件选择为比热解链温度(Tm)低大约30℃。中等严格条件为温度比Tm低20℃，而高严格条件为温度比Tm低10℃。高严格杂交条件通常用于分离与靶核酸序列具有高序列相似性的杂交序列。不过，由于遗传密码的简并性，核酸可以在序列上有偏差而依然编码基本上相同的多肽。因此有时可能需要中等严格杂交条件来鉴定这样的核酸分子。The term "stringency" refers to the conditions under which hybridization occurs. The stringency of hybridization is affected by conditions such as temperature, salt concentration, ionic strength, and hybridization buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Conditions of medium stringency are at temperatures 20°C below the Tm, while conditions of high stringency are at
Tm是在确定的离子强度和pH值时，50％的靶序列与完美匹配的探针杂交的温度。Tm取决于溶液条件和探针的碱基组成及长度。例如，较长的序列在较高温度特异性杂交。在低于Tm值大约16℃到32℃获得最大杂交速率。在杂交溶液中存在一价阳离子会减少两核酸链之间的静电排斥作用，从而促进杂交体形成；当钠浓度不超过0.4M时，这一作用明显(对于更高的浓度，此效应可以忽略不计)。每个百分点的甲酰胺可使DNA-DNA和DNA-RNA双链体的解链温度降低0.6到0.7℃，加入50％甲酰胺能够使杂交在30到45℃进行，尽管这将降低杂交速率。碱基对错配降低杂交速率和双链体的热稳定性。平均而言，对于大的探针，每个百分点碱基错配使Tm值下降约1℃。取决于杂交体的类型，Tm值可以利用下列公式计算：The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe at a defined ionic strength and pH. The Tm depends on the solution conditions and the base composition and length of the probe. For example, longer sequences hybridize specifically at higher temperatures. The maximum rate of hybridization is obtained at about 16°C to 32°C below the Tm value. The presence of monovalent cations in the hybridization solution will reduce the electrostatic repulsion between the two nucleic acid strands, thereby promoting hybrid formation; when the sodium concentration does not exceed 0.4M, this effect is significant (for higher concentrations, this effect can be ignored Excluding). Each percent of formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes by 0.6 to 0.7°C, and adding 50% formamide allows hybridization to proceed at 30 to 45°C, although this will slow down the hybridization rate. Base pair mismatches reduce the rate of hybridization and the thermal stability of the duplex. On average, for large probes, each percent base mismatch reduces the Tm value by about 1°C. Depending on the type of hybrid, the Tm value can be calculated using the following formula:
1)DNA-DNA杂交体(Meinkoth和Wahl，Anal.Biochem.，138：267-284，1984)：1) DNA-DNA hybrid (Meinkoth and Wahl, Anal. Biochem., 138:267-284, 1984):
Tm＝81.5℃+16.6×log10[Na+]a+0.41×％[G/Cb]-500×[Lc]-1-0.61×％甲酰胺Tm＝81.5℃+16.6×log10 [Na+ ]a +0.41×%[G/Cb ]-500×[Lc ]-1 -0.61×% formamide
2)DNA-RNA或RNA-RNA杂交体：2) DNA-RNA or RNA-RNA hybrid:
Tm＝79.8+18.5(log10[Na+]a)+0.58(％G/Cb)+11.8(％G/Cb)2-820/LcTm=79.8+18.5(log10 [Na+ ]a )+0.58(%G/Cb )+11.8(%G/Cb )2 -820/Lc
3)寡DNA或寡RNAd杂交体：3) Oligo DNA or oligo RNAd hybrid:
＜20个核苷酸：Tm＝2(ln)<20 nucleotides: Tm=2(ln )
20-35个核苷酸：Tm＝22+1.46(ln)20-35 nucleotides: Tm=22+1.46(ln )
a或用于其它一价阳离子，但是仅在0.01-0.4M范围内准确。a Or for other monovalent cations, but only accurate in the range of 0.01-0.4M.
b仅对于在30％到75％范围内的％GC是准确的。b is only accurate for %GC in the range of 30% to 75%.
cL＝双链体的碱基对长度。c L = length in base pairs of the duplex.
d寡，寡核苷酸；ln，＝引物的有效长度＝2×(G/C数)+(A/T数)。d oligo, oligonucleotide; ln , =effective length of primer=2×(G/C number)+(A/T number).
非特异性结合可以通过许多已知技术中的任一种来控制，例如用含蛋白质的溶液封闭膜，在杂交缓冲液中添加异源RNA、DNA和SDS，以及用RNA酶处理。对于非同源探针，可以通过改变如下条件之一来进行一系列杂交：(i)逐渐降低退火温度(例如从68℃降至42℃)，或(ii)逐渐降低甲酰胺浓度(例如从50％降至0％)。熟练技术人员知晓可以在杂交过程中改变而保持或者改变严格条件的各种参数。Nonspecific binding can be controlled by any of a number of known techniques, such as blocking membranes with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to hybridization buffer, and treatment with RNases. For non-homologous probes, a series of hybridizations can be performed by changing one of the following conditions: (i) gradually lowering the annealing temperature (e.g., from 68°C to 42°C), or (ii) gradually reducing the formamide concentration (e.g., from 50% down to 0%). The skilled artisan is aware of various parameters that can be altered during hybridization to maintain or alter stringent conditions.
除杂交条件外，杂交特异性通常还是杂交后洗涤的函数。为了除去非特异杂交产生的背景，用稀释的盐溶液洗涤样品。这类洗涤的关键因素包括最终洗涤溶液的离子强度和温度：盐浓度越低、洗涤温度越高，洗涤的严格性就越高。洗涤条件通常在等于或低于杂交严格性的条件下进行。阳性杂交给出至少为背景两倍的信号。一般，适用于核酸杂交测定或基因扩增检测操作的适宜严格条件如上文所示设置。也可以选择更高或更低的严格条件。熟练技术人员知晓可以在洗涤过程中改变从而保持或者改变严格条件的各种参数。In addition to hybridization conditions, hybridization specificity is often a function of post-hybridization washes. To remove background from nonspecific hybridization, samples were washed with dilute saline solution. Key factors in this type of wash include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the more stringent the wash. Washing conditions are generally performed at or below the stringency of hybridization. Positive hybridization gives a signal at least two times background. Generally, suitable stringent conditions for use in nucleic acid hybridization assays or gene amplification detection procedures are set as indicated above. Higher or lower stringency conditions can also be selected. The skilled artisan is aware of various parameters that can be changed during washing to maintain or alter stringent conditions.
例如，长于50个核苷酸的DNA杂交体的典型的高严格杂交条件包括在1×SSC中于65℃杂交或者在1×SSC和50％甲酰胺中于42℃杂交，接着在0.3×SSC中于65℃洗涤。长于50个核苷酸的DNA杂交体的中等严格杂交条件的实例包括在4×SSC中于50℃杂交或者在6×SSC和50％甲酰胺中于40℃杂交，接着在2×SSC中于50℃洗涤。杂交体的长度是杂交核酸的预期长度。当已知序列的核酸进行杂交时，杂交体的长度可以通过比对序列并鉴定本文所述的保守区域来确定。1×SSC是0.15M NaCl和15mM柠檬酸钠；杂交溶液和洗涤溶液可以另外地包括5×Denhardt试剂、0.5-1.0％SDS、100μg/ml片段化的变性鲑精DNA、0.5％焦磷酸钠。For example, typical highly stringent hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 1×SSC at 65° C. or hybridization in 1×SSC and 50% formamide at 42° C., followed by 0.3×SSC Wash at 65°C. Examples of moderately stringent hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 4×SSC at 50° C. or hybridization in 6×SSC and 50% formamide at 40° C., followed by hybridization in 2×SSC at 50° C. Wash at 50°C. The length of the hybrid is the expected length of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the length of the hybrid can be determined by aligning the sequences and identifying conserved regions as described herein. 1 x SSC is 0.15M NaCl and 15 mM sodium citrate; hybridization and wash solutions may additionally include 5 x Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml fragmented denatured salmon sperm DNA, 0.5% sodium pyrophosphate.
为了定义严格性水平，可以参考Sambrook等(2001)的《分子克隆：实验室手册)》，第三版，冷泉港实验室出版，冷泉港，纽约，或者CurrentProtocols in Molecular Biology，John Wiley&Sons，N.Y.(1989及年度更新资料)。To define the level of stringency, reference may be made to Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, New York, or Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. ( 1989 and annual updates).
剪接变体splice variant
本文所用的术语“剪接变体”包括这样的核酸序列变体，其中选择的内含子和/或外显子已被切除、替换、置换或添加，或者其中内含子已被缩短或增长。这样的变体基本上保持了蛋白质的生物活性；这可以通过选择性地保留蛋白质的功能性区段来实现。这样的剪接变体可以是天然的或人工的。预测和分离这类剪接变体的方法是本领域众所周知的(参见例如Foissac和Schiex(2005)BMC Bioinformatics 6：25)。The term "splice variant" as used herein includes nucleic acid sequence variants in which selected introns and/or exons have been excised, substituted, substituted or added, or in which introns have been shortened or lengthened. Such variants substantially retain the biological activity of the protein; this can be achieved by selectively retaining functional segments of the protein. Such splice variants may be natural or artificial. Methods for predicting and isolating such splice variants are well known in the art (see eg Foissac and Schiex (2005) BMC Bioinformatics 6:25).
等位基因变体allelic variant
等位基因或等位基因变体为位于相同的染色体位置的给定基因的可选形式。等位基因变体包括单核苷酸多态性(SNP)，以及小型插入/缺失多态性(INDEL)。INDEL的大小通常小于100bp。在大多数生物体的天然多态性品系中SNP和INDEL形成最大的一组序列变体。Alleles or allelic variants are alternative forms of a given gene that are located at the same chromosomal location. Allelic variants include single nucleotide polymorphisms (SNPs), and small insertion/deletion polymorphisms (INDELs). The size of INDEL is usually less than 100bp. SNPs and INDELs form the largest set of sequence variants in naturally polymorphic strains of most organisms.
基因改组/定向进化Gene shuffling/directed evolution
基因改组或定向进化是重复进行DNA改组以及继之的适当筛选和/或选择，以产生编码具有修饰生物活性的蛋白质的核酸或其部分的变体(Castle等(2004)Science 304(5674)：1151-4；美国专利5,811,238和6,395,547)。Gene shuffling or directed evolution is the repeated practice of DNA shuffling followed by appropriate screening and/or selection to produce variants of nucleic acids or portions thereof encoding proteins with modified biological activity (Castle et al (2004) Science 304(5674): 1151-4; US Patents 5,811,238 and 6,395,547).
调控元件/控制序列/启动子Regulatory elements/control sequences/promoters
术语“调控元件”、“控制序列”和“启动子”在文中均可互换使用，取其广义，是指能够影响与之相连的序列表达的调控性核酸序列。术语“启动子”通常是指位于基因转录起点上游的核酸控制序列，其参与识别和结合RNA聚合酶及其他蛋白质，由此指导有效连接的核酸进行转录。上述术语包括源自经典真核生物基因组基因的转录调控序列(包括对于精确的转录起始是必需的TATA盒，带或不带CCAAT盒序列)，以及其他调控元件(即上游激活序列、增强子和沉默子)——它们通过应答发育刺激和/或外部刺激或以组织特异的方式改变基因表达。该术语还包括经典原核生物基因的转录调控序列，在此情况下可以包括-35盒序列和/或-10盒转录调控序列。术语“调控元件”也涵盖合成的融合分子或衍生物，其赋予、激活或增强细胞、组织或器官中核酸序列分子的表达。The terms "regulatory element", "control sequence" and "promoter" are used interchangeably herein to refer to a regulatory nucleic acid sequence capable of affecting the expression of sequences to which it is linked, in their broadest sense. The term "promoter" generally refers to a nucleic acid control sequence located upstream of the start of gene transcription, which is involved in recognizing and binding RNA polymerase and other proteins, thereby directing the transcription of an operably linked nucleic acid. The above terms include transcriptional regulatory sequences derived from classical eukaryotic genomic genes (including the TATA box necessary for precise transcription initiation, with or without CCAAT box sequences), as well as other regulatory elements (i.e., upstream activating sequences, enhancer and silencers)—they alter gene expression in response to developmental and/or external stimuli or in a tissue-specific manner. The term also includes transcriptional regulatory sequences of classical prokaryotic genes, which in this case may include -35 box sequences and/or -10 box transcriptional regulatory sequences. The term "regulatory element" also covers synthetic fusion molecules or derivatives which confer, activate or enhance the expression of nucleic acid sequence molecules in cells, tissues or organs.
“植物启动子”包括可以介导编码序列区段在植物细胞中表达的调控元件。因此，植物启动子不必是植物来源的，还可来源于病毒或微生物，例如来自攻击植物细胞的病毒。“植物启动子”还可来源于植物细胞，例如，来源于待用欲在本发明方法中表达的以及本文所述的核酸序列转化的植物。这对于其他“植物”调控信号同样适用，例如“植物”终止子。位于可用于本发明方法的核苷酸序列上游的启动子可以通过一个或多个核苷酸取代、插入和/或缺失进行修饰，而不干扰启动子、开放读框(ORF)或者3’调控区如终止子或远离ORF的其他3’调控区的功能或活性。此外，还可以通过修饰启动子的序列而增加其活性，或者将其完全替换为活性更强的启动子、甚至是来自异源生物体的启动子。为在植物中表达，核酸分子必须，如上文所述的那样，有效连接于或者包含适宜的启动子，所述启动子将在恰当的时间点以所需的空间表达模式表达所述基因。A "plant promoter" includes regulatory elements that can mediate the expression of a coding sequence segment in a plant cell. Thus, a plant promoter need not be of plant origin, but may also be derived from viruses or microorganisms, for example from viruses that attack plant cells. A "plant promoter" may also be derived from a plant cell, eg, from a plant to be transformed with a nucleic acid sequence to be expressed in the methods of the invention and described herein. The same applies to other "plant" regulatory signals, such as "plant" terminators. The promoter located upstream of the nucleotide sequence useful in the method of the invention may be modified by one or more nucleotide substitutions, insertions and/or deletions without interfering with the promoter, the open reading frame (ORF) or 3' regulation The function or activity of regions such as terminators or other 3' regulatory regions away from the ORF. In addition, the activity of the promoter can be increased by modifying its sequence, or it can be completely replaced by a more active promoter, even a promoter from a heterologous organism. For expression in plants, the nucleic acid molecule must, as described above, be operably linked to or comprise a suitable promoter which will express the gene at the appropriate point in time with the desired spatial expression pattern.
为鉴定功能上等同的启动子，可以例如通过将候选启动子与报告基因有效连接、测定所述报告基因在植物多种组织中的表达水平和模式，来分析候选启动子的启动子强度和/或表达模式。公知的适宜报告基因包括例如β-葡糖醛酸糖苷酶或β-半乳糖苷酶。通过测量β-葡糖醛酸糖苷酶或β-半乳糖苷酶的酶活来测定启动子活性。然后可以将该启动子强度和/或表达模式与参照启动子(如本发明方法中所用的启动子)相比较。可选地，可以利用本领域公知的方法，如Northern印迹(RNA分析)结合放射自显影图的密度计量分析、定量实时PCR或RT-PCR(Heid等，1996Genome Methods 6：986-994)，通过定量mRNA水平或者将本发明方法所用核酸的mRNA水平与持家基因如18S rRNA的mRNA水平进行比较，来测定启动子强度。通常，“弱启动子”表示驱动编码序列低水平表达的启动子。“低水平”表示每个细胞约1/10,000个转录物到约1/100,000个转录物、到约1/500,0000个转录物的水平。相反，“强启动子”驱动编码序列高水平表达，或者说每个细胞约1/10个转录物到约1/100个转录物、到约1/1000个转录物。一般，“中等强度启动子”表示以低于强启动子的水平，尤其是以在所有情况下都低于在35S CaMV启动子控制下所获得水平的水平，驱动编码序列表达的启动子。To identify functionally equivalent promoters, candidate promoters can be analyzed for promoter strength and/or by, for example, operably linking a candidate promoter to a reporter gene, determining the level and pattern of expression of the reporter gene in various tissues of the plant or expression patterns. Known suitable reporter genes include, for example, beta-glucuronidase or beta-galactosidase. Promoter activity was determined by measuring the enzymatic activity of β-glucuronidase or β-galactosidase. This promoter strength and/or expression pattern can then be compared to a reference promoter, such as the promoter used in the methods of the invention. Alternatively, methods known in the art can be utilized, such as Northern blotting (RNA analysis) combined with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6:986-994), by Promoter strength is determined by quantifying mRNA levels or comparing mRNA levels of nucleic acids used in the methods of the invention to mRNA levels of housekeeping genes such as 18S rRNA. In general, a "weak promoter" refers to a promoter that drives low-level expression of a coding sequence. "Low level" means a level of about 1/10,000 transcript to about 1/100,000 transcript, to about 1/500,0000 transcript per cell. In contrast, a "strong promoter" drives high-level expression of a coding sequence, or about 1/10 transcript to about 1/100 transcript, to about 1/1000 transcript per cell. In general, "moderate strength promoter" means a promoter which drives the expression of a coding sequence at a lower level than that of a strong promoter, especially at a level lower than that obtained under the control of the 35S CaMV promoter in all cases.
有效连接active connection
本文所用的术语“有效连接”是指启动子序列和目的基因之间的功能性连接，从而启动子序列能够起始目的基因的转录。The term "operably linked" as used herein refers to a functional linkage between a promoter sequence and a gene of interest, such that the promoter sequence can initiate transcription of the gene of interest.
组成型启动子constitutive promoter
“组成型启动子”是指在生长和发育的大多数但不必然是所有阶段，在大多数环境条件下，在至少一种细胞、组织或器官中具有转录活性的启动子。下表2a给出了组成型启动子的实例。A "constitutive promoter" refers to a promoter that is transcriptionally active in at least one cell, tissue or organ under most environmental conditions during most, but not necessarily all stages of growth and development. Examples of constitutive promoters are given in Table 2a below.
表2a：组成型启动子的实例Table 2a: Examples of constitutive promoters
遍在启动子ubiquitous promoter
遍在启动子基本上在生物体所有的组织或细胞中都有活性。A ubiquitous promoter is active in essentially all tissues or cells of an organism.
发育调控型启动子developmentally regulated promoter
发育调控型启动子在某些发育阶段或在经历发育改变的植物部分有活性。Developmentally regulated promoters are active at certain developmental stages or in plant parts undergoing developmental changes.
诱导型启动子inducible promoter
诱导型启动子响应化学品(综述参见Gatz 1997，Annu.Rev.PlantPhysiol.Plant Mol.Biol.，48：89-108)、环境或物理刺激而诱导或增加转录起始，或者可以是“胁迫诱导型”，即当植物接触多种胁迫条件时被激活，或者是“病原体诱导型”，即当植物接触多种病原体时被激活。Inducible promoters induce or increase transcription initiation in response to chemical (reviewed in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108), environmental or physical stimuli, or may be "stress-induced "type", which is activated when plants are exposed to multiple stress conditions, or "pathogen-inducible", which is activated when plants are exposed to multiple pathogens.
器官特异性/组织特异性启动子Organ-specific/tissue-specific promoters
器官特异性或组织特异性的启动子是能够在某些器官或组织(如叶、根、种子组织等)中优先起始转录的启动子。例如，“根特异性启动子”是主要在植物根中，基本上排除在植物的任何其他部分中，具有转录活性的启动子，但仍允许在这些其他植物部分中的任何渗漏表达。能够仅在某些细胞中起始转录的启动子在文中称为“细胞特异性”启动子。An organ-specific or tissue-specific promoter is a promoter capable of preferentially initiating transcription in certain organs or tissues (eg, leaves, roots, seed tissues, etc.). For example, a "root-specific promoter" is a promoter that is transcriptionally active primarily in plant roots, to the substantial exclusion of any other parts of the plant, but still allows any leaky expression in these other plant parts. Promoters capable of initiating transcription only in certain cells are referred to herein as "cell-specific" promoters.
根特异性启动子的实例列于下表2b。Examples of root-specific promoters are listed in Table 2b below.
表2b：根特异性启动子的实例Table 2b: Examples of root-specific promoters
种子特异性启动子主要在种子组织中，但不必仅在种子组织中(渗漏表达的情况下)，具有转录活性。种子特异性启动子可以在种子发育和/或萌发期间具有活性。种子特异性启动子可以是胚乳/糊粉层/胚特异性的。种子特异性启动子(胚乳/糊粉层/胚特异性的)的实例列于下表2c至表2f中。种子特异性启动子的更多实例在Qing Qu和Takaiwa(Plant Biotechnol.J.2，113-125，2004)中给出，其公开内容作为参考并入本文，如同充分阐述的那样。A seed-specific promoter is transcriptionally active primarily, but not necessarily exclusively, in seed tissue (in the case of leaky expression). A seed-specific promoter can be active during seed development and/or germination. Seed-specific promoters may be endosperm/aleurone/embryo specific. Examples of seed-specific promoters (endosperm/aleurone/embryo specific) are listed in Tables 2c to 2f below. Further examples of seed-specific promoters are given in Qing Qu and Takaiwa (Plant Biotechnol. J. 2, 113-125, 2004), the disclosure of which is incorporated herein by reference as if fully set forth.
表2c：种子特异性启动子的实例Table 2c: Examples of seed-specific promoters
表2d：胚乳特异性启动子的实例Table 2d: Examples of endosperm-specific promoters
表2e：胚特异性启动子的实例Table 2e: Examples of embryo-specific promoters
表2f：糊粉特异性启动子的实例Table 2f: Examples of aleurone-specific promoters
如文中所定义的绿色组织特异性启动子是主要在绿色组织中，基本上排除在任何其他植物部分中，具有转录活性的启动子，但仍允许在这些其他植物部分中的任何渗漏表达。A green tissue specific promoter as defined herein is a promoter which is transcriptionally active mainly in green tissues, substantially to the exclusion of any other plant parts, but still allowing any leaky expression in these other plant parts.
可以用来实施本发明方法的绿色组织特异性启动子的实例示于下表2g。Examples of green tissue-specific promoters that can be used to practice the methods of the invention are shown in Table 2g below.
表2g：绿色组织特异性启动子的实例Table 2g: Examples of green tissue-specific promoters
组织特异性启动子的另一实例是分生组织特异性启动子，其主要在分生组织中，基本上排除在任何其他植物部分中，具有转录活性，但仍允许在这些其他植物部分的任何渗漏表达。可以用来实施本发明方法的绿色分生组织特异性启动子的实例示于下表2h。Another example of a tissue-specific promoter is a meristem-specific promoter, which is transcriptionally active primarily in the meristems, to the substantial exclusion of any other plant parts, but still permits expression in any of these other plant parts. leaky expression. Examples of green meristem-specific promoters that can be used to practice the methods of the invention are shown in Table 2h below.
表2h：分生组织特异性启动子的实例Table 2h: Examples of meristem-specific promoters
终止子terminator
术语“终止子”包括这样的控制序列，其为位于转录单位末端的DNA序列，发送初级转录物进行3’加工和多聚腺苷酸化以及终止转录的信号。终止子可以源自天然基因、多种其他植物基因、或T-DNA。例如，待加入的终止子可以源自胭脂碱合酶或章鱼碱合酶基因、或可选地源自其它植物基因、或次优选地源自任何其它真核基因。The term "terminator" includes control sequences, which are DNA sequences located at the end of a transcriptional unit, that signal 3' processing and polyadenylation of the primary transcript and termination of transcription. Terminator can be derived from native genes, various other plant genes, or T-DNA. For example, the terminator to be added may be derived from the nopaline synthase or octopine synthase genes, or alternatively from other plant genes, or less preferably from any other eukaryotic gene.
调节adjust
与表达或基因表达相关的术语“调节”是指与对照植物相比，所述基因表达的表达水平被改变的过程，其中表达水平可增加或降低。原始未调节的表达可以是结构RNA(rRNA、tRNA)或随后进行翻译的mRNA的任何类型的表达。术语“调节活性”应理解为本发明核酸序列或编码蛋白质的任何表达改变，该改变导致植物产量增加和/或生长增加。The term "modulation" in relation to expression or gene expression refers to the process by which the expression level of said gene expression is altered compared to control plants, wherein the expression level may be increased or decreased. The original unregulated expression may be any type of expression of structural RNA (rRNA, tRNA) or mRNA that is subsequently translated. The term "modulating activity" is to be understood as any change in the expression of the nucleic acid sequences or encoded proteins according to the invention, which change leads to increased yield and/or increased growth of plants.
表达Express
术语“表达”或“基因表达”是指特定基因或特定基因构建体的转录。术语“表达”或“基因表达”特别地是指基因(一个或多个)或基因构建体至结构RNA(rRNA、tRNA)或mRNA的转录，有或无后者至蛋白质的随后翻译。该过程包括DNA的转录和所获得的mRNA产物的加工。The term "expression" or "gene expression" refers to the transcription of a specific gene or a specific genetic construct. The term "expression" or "gene expression" refers in particular to the transcription of a gene(s) or gene construct into structural RNA (rRNA, tRNA) or mRNA, with or without subsequent translation of the latter into protein. This process involves transcription of DNA and processing of the resulting mRNA product.
增加的表达/过表达Increased expression/overexpression
如本文所用的术语“增加的表达”或“过表达”表示超出原始野生型表达水平的任何形式的表达。The term "increased expression" or "overexpression" as used herein means any form of expression beyond the original wild-type expression level.
增加基因或基因产物表达的方法在本领域有充分的文献记载，且包括，例如由适当的启动子驱动的过表达、转录增强子或翻译增强子的使用。可以将用作启动子或增强子元件的分离的核酸引入非异源形式的多核苷酸的适当位置(一般是上游)，从而上调编码目的多肽的核酸序列的表达。例如，可以通过突变、缺失和/或取代，在体内改变内源启动子(见Kmiec，US5,565,350；Zarling等，WO9322443)，或者可以将分离的启动子在相对于本发明基因的适当方向和距离引入植物细胞中，从而控制基因的表达。Methods of increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by an appropriate promoter, the use of transcriptional or translational enhancers. An isolated nucleic acid for use as a promoter or enhancer element can be introduced into the non-heterologous form of the polynucleotide at an appropriate location (generally upstream) to upregulate expression of a nucleic acid sequence encoding a polypeptide of interest. For example, endogenous promoters can be altered in vivo by mutation, deletion and/or substitution (see Kmiec, US 5,565,350; Zarling et al., WO9322443), or isolated promoters can be oriented and Distance is introduced into plant cells, thereby controlling gene expression.
如果期望多肽表达，通常期望在多核苷酸编码区的3’末端纳入多聚腺苷酸化区域。多聚腺苷酸化区域可以源自天然基因、多种其它植物基因或T-DNA。例如，待加入的3’末端序列可以源自胭脂碱合酶或章鱼碱合酶基因、或可选地源自其他植物基因、或次优选地源自任何其它真核基因。If expression of the polypeptide is desired, it is generally desirable to include a polyadenylation region at the 3' end of the coding region of the polynucleotide. Polyadenylation regions can be derived from native genes, various other plant genes, or T-DNA. For example, the 3' end sequence to be added may be derived from the nopaline synthase or octopine synthase genes, or alternatively from other plant genes, or less preferably from any other eukaryotic gene.
也可以在5’非翻译区(UTR)或部分编码序列的编码序列中加入内含子序列，来增加在胞质中累积的成熟信使的量。已显示，在植物和动物表达构建体的转录单位中纳入可剪接内含子，可以在mRNA和蛋白质水平使基因表达增加高达1000倍(Buchman和Berg(1988)Mol.Cell biol.8：4395-4405；Callis等(1987)Genes Dev.1：1183-1200)。通常内含子放置在转录单位5’末端附近时，增强基因表达的作用最大。玉蜀黍内含子Adh1-S内含子1、2和6，Bronze-1内含子的使用是本领域公知的。一般信息请参见The Maize Handbook，第116章，Freeling和Walbot编辑，Springer，N.Y.(1994)。Intronic sequences can also be added to the coding sequence in the 5' untranslated region (UTR) or part of the coding sequence to increase the amount of mature messenger that accumulates in the cytoplasm. It has been shown that the incorporation of splicable introns in the transcription unit of plant and animal expression constructs can increase gene expression up to 1000-fold at the mRNA and protein levels (Buchman and Berg (1988) Mol. Cell biol. 8: 4395- 4405; Callis et al. (1987) Genes Dev. 1:1183-1200). Usually, when introns are placed near the 5' end of the transcription unit, the effect of enhancing gene expression is greatest. The use of the maize intron Adhl-
内源基因endogenous gene
本文述及“内源”基因不仅指见于植物之中的天然形式的所讨论基因(即未经人为干预)，而且指随后(重新)引入到植物中的分离形式的所述基因(或基本上同源的核酸/基因)(转基因)。例如，含有这样的转基因的转基因植物可遭遇该转基因表达的实质性下降和/或该内源基因表达的实质性下降。该分离的基因可从生物体分离或可以例如通过化学合成进行人造。References herein to an "endogenous" gene refer not only to the gene in question in its natural form (i.e. without human intervention) as found in a plant, but also to an isolated form (or substantially homologous nucleic acid/gene) (transgene). For example, a transgenic plant containing such a transgene may experience a substantial decrease in expression of the transgene and/or a substantial decrease in expression of the endogenous gene. The isolated gene can be isolated from an organism or can be artificial, for example by chemical synthesis.
降低的表达reduced expression
本文述及“降低的表达”或者表达“减小或基本上消除”应理解为表示，内源基因表达和/或多肽水平和/或多肽活性相对于对照植物降低。所述减小或基本上消除按照递增的优选顺序为，与对照植物相比，减小至少10％、20％、30％、40％或50％、60％、70％、80％、85％、90％或95％、96％、97％、98％、99％或更多。用于降低表达的方法在本领域内是已知的，并且本领域技术人员能够容易地通过例如使用适当的启动子来适应性调整用于沉默的已知方法，以实现内源基因在整个植物或植物部分中表达的降低。Reference herein to "reduced expression" or expression "reduced or substantially eliminated" is understood to mean that endogenous gene expression and/or polypeptide level and/or polypeptide activity is reduced relative to control plants. Said reduction or substantial elimination, in increasing order of preference, is at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85% reduction compared to control plants , 90%, or 95%, 96%, 97%, 98%, 99% or more. Methods for reducing expression are known in the art and those skilled in the art can readily adapt known methods for silencing by, for example, using an appropriate promoter, to achieve expression of an endogenous gene throughout the plant. or decreased expression in plant parts.
为减小或基本上消除植物中内源基因的表达，需要一段足够长度的、基本上连续核苷酸的核酸序列。为进行基因沉默，这可以少至20、19、18、17、16、15、14、13、12、11、10或更少的核苷酸，可选地，这可以多至完整的基因(包括部分或完整的5’和/或3’UTR)。此基本上连续的核苷酸链可以源自编码目的蛋白质的核酸(靶基因)，或者源自能够编码目的蛋白质的直向同源物、旁系同源物或同源物的任何核酸。优选地，基本上连续的核苷酸链能够与靶基因(有义链或反义链)形成氢键，更优选地，基本上连续的核苷酸链按照递增的优选顺序与靶基因(有义链或反义链)50％、60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％序列相同。对于本文所讨论的用于减小或基本上消除内源基因表达的各种方法而言，编码(功能性)多肽的核酸序列并非必需的。To reduce or substantially eliminate expression of an endogenous gene in a plant requires a nucleic acid sequence of substantially contiguous nucleotides of sufficient length. For gene silencing this can be as few as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer nucleotides, alternatively this can be as many as the entire gene ( including partial or complete 5' and/or 3'UTR). This substantially continuous chain of nucleotides may be derived from the nucleic acid encoding the protein of interest (the target gene), or from any nucleic acid capable of encoding an orthologue, paralog or homologue of the protein of interest. Preferably, the substantially continuous chain of nucleotides is capable of forming hydrogen bonds with the target gene (sense strand or antisense strand), more preferably, the substantially continuous chain of nucleotides forms hydrogen bonds with the target gene (with Sense strand or antisense strand) 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. A nucleic acid sequence encoding a (functional) polypeptide is not required for the various methods discussed herein for reducing or substantially eliminating expression of an endogenous gene.
用于减小或基本上消除植物中内源基因表达或降低蛋白质的水平和/或活性的多种方法的实例对于本领域技术人员来说是公知的。本领域技术人员能够容易地通过例如使用适当的启动子来适应性调整用于沉默的已知方法，以实现内源基因在整个植物或植物部分中表达的降低。Examples of various methods for reducing or substantially eliminating expression of an endogenous gene or reducing the level and/or activity of a protein in a plant are well known to those skilled in the art. A person skilled in the art can readily adapt known methods for silencing to achieve a reduction in the expression of endogenous genes in whole plants or plant parts, eg by using appropriate promoters.
减小或基本上消除表达可以利用常规工具和技术来实现。减小或基本上消除内源基因表达的一个优选方法是通过向植物中引入和表达基因构建体，其中，核酸(在此情况中，源自目的基因、或者源自能够编码任一目的蛋白质的直向同源物、旁系同源物或同源物的任何核酸的、一段基本上连续核苷酸的链)以被间隔子(非编码DNA)分隔开的、(部分或完全地)反向重复的形式克隆在该构建体中。Reducing or substantially eliminating expression can be achieved using conventional tools and techniques. A preferred method of reducing or substantially eliminating expression of an endogenous gene is by introducing into plants and expressing a genetic construct in which the nucleic acid (in this case, derived from the gene of interest, or from a protein capable of encoding any protein of interest A stretch of substantially contiguous nucleotides) of any nucleic acid of an orthologue, paralogue, or homologue) separated (partially or completely) by spacers (non-coding DNA) An inverted repeat form was cloned in this construct.
在这样的优选方法中，利用核酸或其部分(在此情况中，源自目的基因、或者源自能够编码目的蛋白质的直向同源物、旁系同源物或同源物的任何核酸的、一段基本上连续核苷酸的链)的反向重复(优选能够形成发夹结构)，通过RNA介导的沉默，实现减小或基本上消除内源基因的表达。将该反向重复序列克隆进包含控制序列的表达载体中。非编码DNA核酸序列(间隔子，例如基质附着区片段(MAR)、内含子、多接头等)位于形成该反向重复的两个反向核酸之间。该反向重复序列转录后，形成具有(部分或完全)自我互补结构的嵌合RNA。该双链RNA结构称为发夹RNA(hpRNA)。hpRNA被植物加工成可以整合入RNA诱导的沉默复合物(RISC)中的siRNA。RISC进而切割mRNA转录物，从而显著减少待翻译成多肽的mRNA转录物的数量。关于其他一般细节，参见例如Grierson等(1998)WO 98/53083；Waterhouse等(1999)WO 99/53050)。In such preferred methods, nucleic acid or part thereof (in this case, derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralog or homologue of a protein of interest) is utilized. , a stretch of substantially contiguous nucleotides) (preferably capable of forming a hairpin structure), through RNA-mediated silencing, the reduction or substantial elimination of expression of an endogenous gene is achieved. This inverted repeat was cloned into an expression vector containing control sequences. Non-coding DNA nucleic acid sequences (spacers, such as matrix attachment region segments (MAR), introns, polylinkers, etc.) are located between the two inverted nucleic acids forming the inverted repeat. Upon transcription of this inverted repeat, a chimeric RNA with a (partially or fully) self-complementary structure is formed. This double-stranded RNA structure is called hairpin RNA (hpRNA). hpRNAs are processed by plants into siRNAs that can be incorporated into the RNA-induced silencing complex (RISC). RISC in turn cleaves the mRNA transcript, thereby significantly reducing the number of mRNA transcripts to be translated into polypeptides. For other general details see eg Grierson et al (1998) WO 98/53083; Waterhouse et al (1999) WO 99/53050).
本发明的方法的实施不依赖于向植物中引入和表达其中以反向重复形式克隆了核酸分子的基因构建体，而是可以使用几种公知的“基因沉默”法中的任一个或多个来实现相同的效应。Performance of the methods of the present invention does not rely on the introduction and expression into plants of a genetic construct in which the nucleic acid molecule is cloned as an inverted repeat, but may use any one or more of several well-known "gene silencing" methods to achieve the same effect.
用于减小内源基因表达的一个这样的方法是RNA介导的基因表达的沉默(下调)。在该情况下沉默在植物中由双链RNA序列(dsRNA)触发，所述双链RNA序列基本上与靶内源基因相似。该dsRNA被植物进一步加工成称为短干扰RNA(siRNA)的大约20至大约26个核苷酸。siRNA整合入RNA诱导的沉默复合物(RISC)，该复合物切割内源靶基因的mRNA转录物，从而实质性减少待翻译成多肽的mRNA转录物的数量。优选，双链RNA序列相应于靶基因。One such method for reducing expression of endogenous genes is RNA-mediated silencing (downregulation) of gene expression. In this case silencing is triggered in plants by a double-stranded RNA sequence (dsRNA) which is substantially similar to the target endogenous gene. This dsRNA is further processed by the plant into about 20 to about 26 nucleotides called short interfering RNA (siRNA). The siRNA incorporates into the RNA-induced silencing complex (RISC), which cleaves the mRNA transcript of the endogenous target gene, thereby substantially reducing the amount of the mRNA transcript to be translated into a polypeptide. Preferably, the double-stranded RNA sequence corresponds to the target gene.
RNA沉默法的另一实例包括以有义取向，向植物中引入核酸序列或其部分(在这种情况下，源自目的基因、或者源自能够编码目的蛋白质的直向同源物、旁系同源物或同源物的任何核酸的、一段基本上连续核苷酸的链)。“有义取向”是指与其mRNA转录物同源的DNA序列。从而至少一个拷贝的核酸序列被引入植物。该额外的核酸序列将减小内源基因的表达，从而产生称为共抑制的现象。如果将几个额外拷贝的核酸序列引入植物，则基因表达的减小将更明显，因为在高转录水平与共抑制的触发之间存在正相关。Another example of an RNA silencing method involves introducing into a plant a nucleic acid sequence or part thereof (in this case derived from a gene of interest, or from an orthologue, paralog, or paralog capable of encoding a protein of interest) in a sense orientation. A homologue or a stretch of substantially contiguous nucleotides of any nucleic acid of a homologue). "Sense orientation"refers to DNA sequences that are homologous to their mRNA transcripts. Thereby at least one copy of the nucleic acid sequence is introduced into the plant. This additional nucleic acid sequence will reduce the expression of the endogenous gene, thereby producing a phenomenon known as co-suppression. If several extra copies of the nucleic acid sequence are introduced into the plant, the reduction in gene expression will be more pronounced, since there is a positive correlation between high transcription levels and the triggering of co-suppression.
RNA沉默法的另一实例包括使用反义核酸序列。“反义”核酸序列包含这样的核苷酸序列，所述核苷酸序列与编码蛋白质的“有义”核酸序列互补，即与双链cDNA分子的编码链互补或与mRNA转录物序列互补。反义核酸序列优选与待沉默的内源基因互补。互补性可位于基因的“编码区”和/或“非编码区”中。术语“编码区”是指包含将翻译成氨基酸残基的密码子的核苷酸序列的区域。术语“非编码区”是指连接在编码区侧翼的5′和3′序列，其可被转录但不被翻译成氨基酸(也称为5′和3′非翻译区)。Another example of RNA silencing methods involves the use of antisense nucleic acid sequences. An "antisense" nucleic acid sequence comprises a nucleotide sequence that is complementary to a protein-encoding "sense" nucleic acid sequence, ie, to the coding strand of a double-stranded cDNA molecule or to an mRNA transcript sequence. The antisense nucleic acid sequence is preferably complementary to the endogenous gene to be silenced. Complementarity can be located in "coding regions" and/or "non-coding regions" of a gene. The term "coding region" refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues. The term "non-coding region" refers to the 5' and 3' sequences flanking the coding region, which are transcribed but not translated into amino acids (also called 5' and 3' untranslated regions).
可根据沃尔森和克里克碱基配对法则设计反义核酸序列。反义核酸序列可与整个核酸序列(在这种情况下，源自目的基因、或者源自能够编码目的蛋白质的直向同源物、旁系同源物或同源物的任何核酸的、一段基本上连续核苷酸的链)互补，但也可以是仅对核酸序列的部分(包括mRNA 5’和3’UTR)反义的寡核苷酸。例如，反义寡核苷酸序列可与围绕编码多肽的mRNA转录物的翻译起始位点的区域互补。适宜的反义寡核苷酸序列的长度在本领域内是已知的并且可以开始于长大约50、45、40、35、30、25、20、15或10个核苷酸或更少。可使用本领域内已知的方法，使用化学合成和酶促连接反应，构建根据本发明的反义核酸序列。例如，反义核酸序列(例如，反义寡核苷酸序列)可使用天然存在的核苷酸或各种修饰核苷酸来化学合成，所述修饰核苷酸经设计用以增加分子的生物学稳定性或增加反义与有义核酸序列之间形成的双链体的物理稳定性，例如可使用硫代磷酸酯衍生物和吖啶取代的核苷酸。可用于产生反义核酸序列的修饰核苷酸的实例在本领域是公知的。已知的核苷酸修饰包括甲基化、环化和“加帽”和用类似物例如肌苷对一个或多个天然存在的核苷酸的取代。核苷酸的其他修饰在本领域是公知的。Antisense nucleic acid sequences can be designed according to the rules of Walson and Crick base pairing. The antisense nucleic acid sequence can be combined with the entire nucleic acid sequence (in this case, a segment, a stretch, or a segment derived from any nucleic acid that is capable of encoding an orthologue, paralogue, or homolog of a protein of interest) A strand of essentially contiguous nucleotides), but can also be an oligonucleotide that is antisense to only a portion of a nucleic acid sequence (including the mRNA 5' and 3' UTR). For example, the antisense oligonucleotide sequence can be complementary to a region surrounding the translation initiation site of an mRNA transcript encoding a polypeptide. The length of suitable antisense oligonucleotide sequences is known in the art and may start at about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides or less in length. Antisense nucleic acid sequences according to the invention can be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art. For example, antisense nucleic acid sequences (e.g., antisense oligonucleotide sequences) can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological To increase the chemical stability or increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, for example phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides that can be used to generate antisense nucleic acid sequences are well known in the art. Known nucleotide modifications include methylation, cyclization, and "capping" and substitution of one or more naturally occurring nucleotides with analogs such as inosine. Other modifications of nucleotides are well known in the art.
可使用已将核酸序列以反义取向(即，从插入的核酸转录的RNA针对目的靶核酸是反义取向)亚克隆入其中的表达载体，生物学地产生反义核酸序列。优选，植物中，通过稳定地整合的包含启动子、有效连接的反义寡核苷酸和终止子的核酸构建体，产生反义核酸序列。Antisense nucleic acid sequences can be produced biologically using expression vectors into which the nucleic acid sequences have been subcloned in an antisense orientation (ie, RNA transcribed from an inserted nucleic acid is in an antisense orientation to a target nucleic acid of interest). Preferably, the antisense nucleic acid sequence is produced in plants by a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide and a terminator.
用于在本发明的方法中进行沉默的核酸分子(无论引入植物的还是原位产生的)与编码多肽的mRNA转录物和/或基因组DNA杂交或结合，从而例如通过抑制转录和/或翻译来抑制蛋白质的表达。杂交可通过常规核苷酸互补性以形成稳定的双链体或者，例如在结合DNA双链体的反义核酸序列的情况下，通过双螺旋的大沟中的特定相互作用而产生。可通过转化或在特定组织位置直接注射，将反义核酸序列引入植物。可选地，可修饰反义核酸序列以靶向选择的细胞，然后全身性施用。例如，为了进行全身性施用，可以修饰反义核酸序列，以便其特异性结合选择的细胞表面上表达的受体或抗原(例如，通过将反义核酸序列连接至结合细胞表面受体或抗原的肽或抗体)。还可使用本文中描述的载体将反义核酸序列递送至细胞。The nucleic acid molecule used for silencing in the methods of the invention (whether introduced into a plant or produced in situ) hybridizes or binds to an mRNA transcript encoding a polypeptide and/or to genomic DNA, thereby for example by inhibiting transcription and/or translation Inhibit protein expression. Hybridization may occur by conventional nucleotide complementarity to form stable duplexes or, eg, in the case of antisense nucleic acid sequences that bind DNA duplexes, by specific interactions in the major groove of the double helix. Antisense nucleic acid sequences can be introduced into plants by transformation or direct injection at specific tissue locations. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically. For example, for systemic administration, an antisense nucleic acid sequence can be modified so that it specifically binds a receptor or antigen expressed on the surface of a selected cell (e.g., by linking the antisense nucleic acid sequence to a receptor or antigen that binds a cell surface receptor or antigen). peptide or antibody). Antisense nucleic acid sequences can also be delivered to cells using the vectors described herein.
根据另一个方面，反义核酸序列是α-异头物核酸序列。α-异头物核酸序列与互补RNA形成特定的双链杂交体，其中与常见的b单元(b-units)不同，链走向彼此平行(Gaultier等(1987)Nucl Ac Res 15：6625-6641)。反义核酸序列还可包含2′-o-甲基核糖核苷酸(Inoue等(1987)Nucl Ac Res15，6131-6148)或嵌合RNA-DNA类似物(Inoue等(1987)FEBS Lett.215，327-330)。According to another aspect, the antisense nucleic acid sequence is an alpha-anomeric nucleic acid sequence. The α-anomeric nucleic acid sequence forms specific double-stranded hybrids with complementary RNA in which, unlike the common b-units (b-units), the strands run parallel to each other (Gaultier et al. (1987) Nucl Ac Res 15:6625-6641) . The antisense nucleic acid sequence can also comprise 2'-o-methyl ribonucleotides (Inoue et al. (1987) Nucl Ac Res15, 6131-6148) or chimeric RNA-DNA analogs (Inoue et al. , 327-330).
还可使用核酶减少或基本上消除内源基因的表达。核酶是具有核糖核酸酶活性的催化性RNA分子，该分子能够切割与其具有互补区的单链核酸序列例如mRNA。因此，核酶(例如，锤头核酶(Haselhoff和Gerlach(1988)Nature 334，585-591)中描述的)可用于催化切割编码多肽的mRNA转录物，从而显著减少待翻译成多肽的mRNA的数量。可设计具有对于核酸序列的特异性的核酶(参见例如：Cech等美国专利号4,987,071；和Cech等美国专利号5,116,742)。可选择地，可以使用相应于核酸序列的mRNA转录物，从RNA分子库中选择具有特定核糖核酸酶活性的催化性RNA(Bartel和Szostak(1993)Science 261，1411-1418)。核酶用于在植物中进行基因沉默的用途在本领域是已知的(例如，Atkins等(1994)WO 94/00012；Lenne等(1995)WO 95/03404；Lutziger等(2000)WO 00/00619；Prinsen等(1997)WO 97/13865和Scott等(1997)WO 97/38116)。Ribozymes can also be used to reduce or substantially eliminate expression of endogenous genes. A ribozyme is a catalytic RNA molecule with ribonuclease activity capable of cleaving a single-stranded nucleic acid sequence, such as mRNA, to which it has a complementary region. Thus, ribozymes such as those described in Hammerhead ribozyme (Haselhoff and Gerlach (1988) Nature 334, 585-591 ) can be used to catalytically cleave mRNA transcripts encoding polypeptides, thereby significantly reducing the amount of mRNA to be translated into polypeptides. quantity. Ribozymes can be designed with specificity for nucleic acid sequences (see, eg, Cech et al., US Patent No. 4,987,071; and Cech et al., US Patent No. 5,116,742). Alternatively, catalytic RNAs with specific ribonuclease activity can be selected from a library of RNA molecules using mRNA transcripts corresponding to the nucleic acid sequences (Bartel and Szostak (1993) Science 261, 1411-1418). The use of ribozymes for gene silencing in plants is known in the art (e.g., Atkins et al (1994) WO 94/00012; Lenne et al (1995) WO 95/03404; Lutziger et al (2000) WO 00/ 00619; Prinsen et al. (1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).
基因沉默还可以通过插入诱变(例如，T-DNA插入或转座子插入)或通过Angell和Baulcombe((1999)Plant J 20(3)：357-62)、(Amplicon VIGSWO 98/36083)或Baulcombe(WO 99/15682)等所述的策略来实现。Gene silencing can also be achieved by insertional mutagenesis (e.g., T-DNA insertion or transposon insertion) or by Angell and Baulcombe ((1999) Plant J 20(3):357-62), (
如果在内源基因上存在突变和/或在随后引入植物的分离基因/核酸上存在突变，那么基因沉默也可发生。减少或基本上消除可通过非功能性多肽引起。例如，多肽可能结合多种相互作用的蛋白质；因此，可以通过一个或多个突变和/或截短，提供仍然能够结合相互作用的蛋白质(例如受体蛋白)但不能展示其正常功能(例如信号转导配体)的多肽。Gene silencing can also occur if there are mutations in the endogenous gene and/or in an isolated gene/nucleic acid that is subsequently introduced into the plant. Reduction or substantial elimination can be caused by non-functional polypeptides. For example, a polypeptide may bind multiple interacting proteins; thus, one or more mutations and/or truncations may be provided that are still capable of binding interacting proteins (e.g. receptor proteins) but are unable to exhibit their normal function (e.g. signaling transduction ligand) polypeptide.
进行基因沉默的另一个方法是通过用与基因的调控区(例如启动子和/或增强子)互补的核酸序列来打靶以形成三螺旋结构，所述结构阻止基因在靶细胞中的转录。参见Helene，C.，Anticancer Drug Res.6，569-84，1991；Helene等，Ann.N.Y.Acad.Sci.660，27-361992；和Maher，L.J.Bioassays14，807-15，1992。Another method of gene silencing is by targeting with nucleic acid sequences that are complementary to the regulatory regions of the gene (eg, the promoter and/or enhancer) to form triple-helical structures that prevent transcription of the gene in target cells. See Helene, C., Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N. Y. Acad. Sci. 660, 27-36 1992;
其他方法，例如应用针对内源多肽的抗体在植物原位(in planta)抑制其功能、或干扰多肽所参与的信号传递通路，对于技术人员是公知的。特别地，可预期人造分子可用于抑制靶多肽的生物功能，或用于干扰其中靶多肽参与的信号转导途径。Other methods, such as the use of antibodies against endogenous polypeptides to inhibit their function in planta (in planta), or interfere with signal transduction pathways in which the polypeptides participate, are well known to the skilled person. In particular, it is contemplated that the artificial molecule can be used to inhibit the biological function of the target polypeptide, or to interfere with a signal transduction pathway in which the target polypeptide participates.
可选择地，可设置筛选程序以鉴定植物群体中基因的天然变体，该变体编码具有减少的活性的多肽。这样的天然变体也可用于例如进行同源重组。Alternatively, screening programs can be set up to identify natural variants of genes in plant populations that encode polypeptides with reduced activity. Such natural variants may also be used, for example, to perform homologous recombination.
人工和/或天然微小RNA(miRNA)可以用来敲除基因表达和/或mRNA翻译。内源miRNA为单链小RNA，一般长度19-24个核苷酸。它们主要用于调控基因表达和/或mRNA翻译。大多数植物microRNA(miRNA)具有与其靶序列完全或几乎完全的互补性。然而，存在具有达到5个错配的天然靶。miRNA利用Dicer家族的双链特异性RNA酶从具有特征性折回结构的更长的非编码RNA加工而来。一旦加工后，它们通过结合RNA诱导的沉默复合物(RISC)的主要成分Argonaute蛋白，而掺入到RNA诱导沉默复合物中。MiRNA充当RISC的特异性组件，因为它们与细胞质中的靶核酸(大多数为mRNA)碱基配对。随后的调控事件包括靶mRNA切割和破坏和/或翻译抑制。因此，miRNA过表达的效应常反映为靶基因的降低的mRNA水平。Artificial and/or natural microRNAs (miRNAs) can be used to knock down gene expression and/or mRNA translation. Endogenous miRNA is a single-stranded small RNA, generally 19-24 nucleotides in length. They are primarily used to regulate gene expression and/or mRNA translation. Most plant microRNAs (miRNAs) have complete or almost complete complementarity to their target sequences. However, there are natural targets with up to 5 mismatches. miRNAs are processed from longer noncoding RNAs with characteristic snapback structures using double-strand-specific RNases of the Dicer family. Once processed, they are incorporated into the RNA-induced silencing complex (RISC) by binding to the Argonaute proteins, major components of the RNA-induced silencing complex (RISC). MiRNAs act as specific components of RISC because they base pair with target nucleic acids (mostly mRNAs) in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational repression. Thus, the effects of miRNA overexpression are often reflected in reduced mRNA levels of target genes.
人工微小RNA(amiRNA)一般长度21个核苷酸，可以特异地遗传改造以负调控单个或多个目的基因的基因表达。植物微小RNA靶标选择的决定因素在本领域公知。已经定义了靶标识别的经验参数，并且可用来辅助设计特异性amiRNA(Schwab等，(2005)Dev Cell 8：517-527，2005)。设计和生成amiRNA及其前体的便利工具也是公众可获得的(Schwab等，(2006)Plant Cell 18(5)：1121-1133，2006)。Artificial microRNA (amiRNA) is generally 21 nucleotides in length and can be specifically genetically engineered to negatively regulate gene expression of single or multiple target genes. The determinants of plant microRNA target selection are well known in the art. Empirical parameters for target recognition have been defined and can be used to aid in the design of specific amiRNAs (Schwab et al. (2005) Dev Cell 8:517-527, 2005). Convenient tools for designing and generating amiRNAs and their precursors are also publicly available (Schwab et al., (2006) Plant Cell 18(5):1121-1133, 2006).
为优化性能，用来减小植物中内源基因表达的基因沉默技术需要应用来自单子叶植物的核酸序列转化单子叶植物，而使用来自双子叶植物的核酸序列转化双子叶植物。优选，将来自任何给定植物物种的核酸序列引入到相同物种中。例如，来自稻的核酸序列转化到稻植物中。然而，待引入的核酸序列来源于与其待引入的植物相同的植物物种并非是绝对必需的。内源靶基因与待引入的核酸之间基本上同源就足够了。For optimal performance, gene silencing techniques for reducing expression of endogenous genes in plants require transformation of monocotyledonous plants with nucleic acid sequences from monocotyledonous plants and transformation of dicotyledonous plants with nucleic acid sequences from dicotyledonous plants. Preferably, a nucleic acid sequence from any given plant species is introduced into the same species. For example, a nucleic acid sequence from rice is transformed into a rice plant. However, it is not absolutely necessary that the nucleic acid sequence to be introduced originates from the same plant species as the plant into which it is to be introduced. Substantial homology between the endogenous target gene and the nucleic acid to be introduced is sufficient.
上文描述了减小或基本上消除植物中内源基因表达的多种方法的实例。本领域技术人员将能够容易地调整上述沉默方法，以便例如通过应用适当的启动子而实现内源基因在整株植物或其部分中的表达减小。Examples of various methods for reducing or substantially eliminating expression of endogenous genes in plants are described above. A person skilled in the art will readily be able to adapt the above-described silencing methods to achieve reduced expression of the endogenous gene in the whole plant or in parts thereof, for example by applying an appropriate promoter.
可选择标记(基因)/报告基因Selectable marker (gene)/reporter gene
“可选择标记”、“可选择标记基因”或“报告基因”包括赋予细胞表型的任何基因，其中该表型在细胞中的表达有利于鉴定和/或选择经本发明的核酸构建体转染或转化的细胞。这些标记基因通过一系列不同的原理使得能够鉴定核酸分子的成功转移。适宜的标记可以选自赋予抗生素或除草剂抗性、引入新的代谢性状或允许可视选择的标记。可选择标记基因的实例包括赋予抗生素抗性的基因(例如磷酸化新霉素和卡那霉素的nptII，或磷酸化潮霉素的hpt，或赋予抗例如博来霉素、链霉素、四环素、氯霉素、氨苄青霉素、庆大霉素、遗传霉素(G418)、壮观霉素或杀稻瘟素抗性的基因)、赋予除草剂抗性的基因(例如提供抗
已知对于核酸在植物细胞中的稳定或瞬时整合，取决于所用的表达载体和所用的转染技术，仅少数细胞可以摄入该外来DNA，以及，如果期望的话，整合进其基因组。为鉴定并选择这些整合体，通常将编码可选择标记(例如上文所述的那些)的基因与目的基因一起引入宿主细胞中。这些标记能够在例如突变体中使用，所述突变体中原有的这些基因例如通过常规方法缺失而没有功能。此外，编码可选择标记的核酸分子可与编码本发明多肽的或用于本发明方法的序列包含在同一个载体中，或者在分开的载体中引入宿主细胞。已经稳定转染了所引入的核酸的细胞可以例如通过选择(例如，整合有可选择标记的细胞存活而其他细胞死去)予以鉴定。一旦不再需要标记基因，可从转基因细胞除去或切除其。用于标记基因去除的技术在本领域是已知的，有用的技术在上文中描述于定义部分。It is known that for stable or transient integration of nucleic acids in plant cells, depending on the expression vector used and the transfection technique used, only a few cells can take up this foreign DNA and, if desired, integrate into its genome. To identify and select for these integrants, typically a gene encoding a selectable marker (such as those described above) is introduced into the host cell along with the gene of interest. These markers can be used, for example, in mutants in which these genes have been deleted without function, for example by conventional methods. Furthermore, a nucleic acid molecule encoding a selectable marker can be included in the same vector as a sequence encoding a polypeptide of the invention or used in a method of the invention, or introduced into a host cell in a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified, eg, by selection (eg, cells incorporating the selectable marker survive while other cells die). Once the marker gene is no longer needed, it can be removed or excised from the transgenic cell. Techniques for marker gene removal are known in the art, useful techniques are described above in the Definitions section.
由于一旦成功引入了核酸后将不再需要或不期望转基因宿主细胞中存在标记基因，特别是抗生素和除草剂抗性基因，所以根据本发明用于引入核酸的方法最好采用能够除去或切除这些标记基因的技术。一种这样的方法是称为共转化的方法。共转化法采用两个载体同时进行转化，一个载体携带根据本发明的核酸，而第二个携带标记基因。很大比例的转化体接收或者对于植物而言(高达40％或以上的转化体)含有两个载体。对于农杆菌转化，转化体通常只接收载体的一部分，即被T-DNA侧翼包围的序列，其通常是表达盒。随后可通过杂交从转化植物中除去标记基因。在另一种方法中，利用整合在转座子中的标记基因与期望的核酸一起进行转化(称为Ac/Ds技术)。转化体可与转座酶来源杂交，或者用赋予转座酶表达的核酸构建体来瞬时或稳定转化转化体。在有些情况下(约10％)，一旦成功进行了转化，转座子会跳离宿主细胞基因组并丢失。在另外一些情况下，转座子会跳至不同的位置。在这些情况下，必须通过杂交以消除标记基因。在微生物学领域，已经研发了使得可以或便于检测此类事件的技术。另一有利的方法有赖于所谓的重组系统；其优势在于可以免除杂交消除。最著名的这类系统是称为Cre/lox系统的系统。Cre1为重组酶，其切除位于loxP序列之间的序列。如果标记基因整合在loxP序列之间，一旦转化成功后，其会因Cre1重组酶的表达而得以切除。其他重组系统有HIN/HIX、FLP/FRT和REP/STB系统(Tribble等，J.Biol.Chem.，275，2000：22255-22267；Velmurugan等，J.Cell Biol.，149，2000：553-566)。根据本发明的核酸序列可以位点特异性地整合进植物基因组。这些方法自然也可以应用于微生物如酵母、真菌或细菌。Since marker genes, in particular antibiotic and herbicide resistance genes, are no longer required or expected to be present in the transgenic host cell once the nucleic acid has been successfully introduced, methods for introducing nucleic acids according to the invention preferably employ methods that remove or excise these genes. Techniques for marker genes. One such method is a method known as co-transformation. The co-transformation method uses two vectors for simultaneous transformation, one carrying the nucleic acid according to the invention and the second carrying the marker gene. A large proportion of transformants received or contained both vectors for plants (up to 40% or more of transformants). For Agrobacterium transformation, the transformants usually receive only a part of the vector, ie the sequence flanked by the T-DNA, which is usually the expression cassette. The marker gene can subsequently be removed from transformed plants by crossing. In another approach, a marker gene integrated in a transposon is used for transformation together with the desired nucleic acid (referred to as Ac/Ds technology). Transformants can be hybridized to a source of transposase, or transformed transiently or stably with a nucleic acid construct that confers expression of the transposase. In some cases (approximately 10%), once transformation has been successfully performed, the transposon jumps out of the host cell genome and is lost. In other cases, the transposon jumps to a different location. In these cases, marker genes must be eliminated by crossing. In the field of microbiology, techniques have been developed that allow or facilitate the detection of such events. Another advantageous approach relies on the so-called recombination system; its advantage is that elimination of hybridization can be dispensed with. The most famous system of this type is that known as the Cre/lox system. Cre1 is a recombinase that excises sequences located between loxP sequences. If the marker gene is integrated between the loxP sequences, it will be excised by the expression of Cre1 recombinase once the transformation is successful. Other recombination systems are HIN/HIX, FLP/FRT and REP/STB systems (Tribble et al., J.Biol.Chem., 275, 2000:22255-22267; Velmurugan et al., J.Cell Biol., 149, 2000:553- 566). The nucleic acid sequences according to the invention can be integrated site-specifically into the plant genome. These methods can naturally also be applied to microorganisms such as yeasts, fungi or bacteria.
转基因的/转基因/重组transgenic/transgenic/recombinant
出于本发明的目的，就例如本发明的核酸序列、含有所述核酸序列的表达盒、基因构建体或载体、或用所述核酸序列、表达盒或载体转化的生物体而言，“转基因的”、“转基因”或“重组”是指所有这些构建体通过重组方法产生，其中：For the purposes of the present invention, "transgenic" refers to, for example, a nucleic acid sequence of the present invention, an expression cassette containing said nucleic acid sequence, a genetic construct or a vector, or an organism transformed with said nucleic acid sequence, expression cassette or vector. ", "transgenic" or "recombinant" means that all such constructs are produced by recombinant means, wherein:
(a)编码可用于本发明方法的蛋白质的核酸序列，或(a) a nucleic acid sequence encoding a protein that can be used in the methods of the invention, or
(b)有效连接于本发明核酸序列的遗传控制序列，例如启动子，或(b) a genetic control sequence, such as a promoter, operably linked to the nucleic acid sequence of the present invention, or
(c)(a)和(b)(c)(a) and (b)
不存在于其天然遗传环境中，或者已通过重组方法修饰，该修饰可以采取的形式为例如一个或多个核苷酸残基的取代、添加、缺失、倒位或插入。天然遗传环境应理解为原始植物中天然的基因组或染色体座位或者存在于基因组文库之中。在基因组文库的情况下，优选保持、至少是部分地保持核酸序列的天然遗传环境。该环境至少位于核酸序列的一侧，长度至少为50bp、优选至少500bp、特别优选至少1000bp、最优选至少5000bp。当天然存在的表达盒——例如编码可用于本发明方法的多肽的相应核酸序列与该核酸序列的天然启动子之间的天然组合——经非天然的合成(“人工”)方法例如诱变处理而被修饰时，此表达盒变成转基因表达盒。合适的方法描述在例如，US 5,565,350或WO 00/15815中。Not present in its natural genetic environment, or has been modified by recombinant means, which modification may take the form, for example, of a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment is understood to mean the genome or chromosomal locus that is native to the original plant or exists in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably maintained, at least partially maintained. The environment is located at least on one side of the nucleic acid sequence, and has a length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, most preferably at least 5000 bp. When a naturally occurring expression cassette—for example, the natural combination between a corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the invention and a natural promoter for the nucleic acid sequence—is subjected to non-natural synthetic ("artificial") methods such as mutagenesis When modified by manipulation, this expression cassette becomes a transgenic expression cassette. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815.
因此，如上文所述，用于本发明目的的转基因植物应理解为指：在所述植物的基因组中，本发明方法中所用的核酸不在其天然基因座上，其中所述核酸可以进行同源或异源表达。不过，正如所提到的那样，转基因也表示：尽管在植物基因组中根据本发明的或本发明方法中所用的核酸在其天然位置上，但是所述序列已相对于天然序列而被修饰，和/或天然序列的调控序列已被修饰。转基因优选理解为表示：根据本发明的核酸在基因组中非天然的座位上表达，即同源表达，或者优选发生核酸的异源表达。优选的转基因植物在文中述及。Therefore, as stated above, a transgenic plant for the purposes of the present invention is understood to mean that in the genome of said plant the nucleic acid used in the method according to the invention is not located at its natural locus, wherein said nucleic acid can be homologous or heterologous expression. However, as mentioned, transgenic also means that although the nucleic acid according to the invention or used in the method according to the invention is in its natural position in the plant genome, said sequence has been modified relative to the native sequence, and and/or the regulatory sequences of the native sequence have been modified. Transgenic is preferably understood to mean that the nucleic acid according to the invention is expressed at an unnatural locus in the genome, ie homologous expression, or preferably heterologous expression of the nucleic acid takes place. Preferred transgenic plants are described herein.
转化convert
本文述及的术语“引入”或“转化”包括将外源多核苷酸转移进宿主细胞，不考虑转移所用的方法。能够随后通过器官发生或者胚胎发生进行克隆增殖的植物组织都可以使用本发明的遗传构建体转化，并从其再生整个植物。具体的组织选择将因可用于和最适于待转化的具体物种的克隆增殖系统而变。示例性的组织靶标包括叶盘、花粉、胚、子叶、下胚轴、雌配子、愈伤组织、既有的分生组织(例如顶端分生组织、腋芽和根分生组织)，以及诱导的分生组织(例如子叶分生组织和下胚轴分生组织)。可以将多核苷酸瞬时地或稳定地引入宿主细胞，并且可以，例如作为质粒以非整合的状态维持。可选地，其可以整合进入宿主基因组。得到的转化植物细胞可以接着以本领域技术人员已知的方式再生为转化的植物。The term "introducing" or "transforming" as used herein includes the transfer of exogenous polynucleotides into a host cell, regardless of the method used for the transfer. Plant tissues capable of subsequent clonal propagation by organogenesis or embryogenesis can be transformed with the genetic constructs of the invention and whole plants regenerated therefrom. The particular tissue choice will vary with the clonal propagation systems available and most suitable for the particular species to be transformed. Exemplary tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, female gametes, callus, established meristems (e.g., apical meristems, axillary buds, and root meristems), and induced Meristems (eg cotyledon meristem and hypocotyl meristem). A polynucleotide can be introduced into a host cell transiently or stably, and can be maintained in a non-integrated state, eg, as a plasmid. Alternatively, it can integrate into the host genome. The resulting transformed plant cells can then be regenerated into transformed plants in a manner known to those skilled in the art.
外来基因转移进入植物基因组中称为转化。植物物种的转化目前是一种相当常规的技术。有利地，可以使用若干转化方法的任一种向适当的祖先细胞引入目的基因。可以利用公开的转化方法以及由植物组织或植物细胞再生植物的方法来进行瞬时或稳定转化。转化方法包括应用脂质体、电穿孔、增加游离DNA摄取的化学物质、直接向植物注射DNA、粒子枪轰击、用病毒或花粉转化和微粒轰击。方法可以选自用于原生质体的钙/聚乙二醇方法(Krens，F.A.等，(1882)Nature 296，72-74；Negrutiu I.等，(1987)Plant Mol.Biol.8：363-373)；原生质体的电穿孔法(Shillito R.D.等，(1985)Bio/Technol 3，1099-1102)；植物材料的显微注射(Crossway A.等，(1986)Mol.Gen Genet 202：179-185)；DNA或RNA包被的粒子轰击(Klein T.M.等，(1987)Nature 327：70)；用(非整合型)病毒感染，等等。优选通过农杆菌介导的转化，产生转基因植物，包括转基因作物植物。有利的转化法是植物原位转化。为此，可以例如使农杆菌作用于植物种子，或用农杆菌接种植物分生组织。已经证明，根据本发明尤为有利的是使转化的农杆菌悬液作用于完整植株或至少花原基。随后培养植物，直至获得所处理植物的种子(Clough和Bent，Plant J.(1998)16，735-743)。农杆菌介导的稻转化方法包括公知的稻转化方法，例如在任一如下文献中描述的那些：欧洲专利申请EP 1198985A1，Aldemita和Hodges(Planta，199：612-617，1996)；Chan等(Plant Mol.Biol.22(3)491-506，1993)，Hiei等(Plant J.6(2)：271-282，1994)，其公开内容并入本文作为参考，如同充分阐述的那样。至于玉米转化，优选的方法如Ishida等(Nat.Biotechnol.14(6)：745-50，1996)或Frame等(Plant Physiol.129(1)：13-22，2002)中所述，其公开内容并入本文作为参考，如同充分阐述的那样。作为举例说明，所述方法还由B.Jenes等，Techniques for Gene Transfer，在Transgenic Plants，卷1，Engineeringand Utilization，编辑S.D.Kung和R.Wu，Academic Press(1993)128-143以及Potrykus Annu.Rev.Plant Physiol.Plant Molec.Biol.42(1991)205-225)中进一步描述。优选将待表达的核酸或构建体克隆到载体中，所述载体适用于转化根癌农杆菌(Agrobacterium tumefaciens)，例如pBin19(Bevan等，Nucl.Acids Res.12(1984)8711)。然后以已知的方式利用由这样的载体转化的农杆菌来转化植物，例如模式植物，像拟南芥属植物(拟南芥(Arabidopsis thaliana)在本发明范围内不视为作物植物)；或者作物植物，例如烟草植物，例如通过将擦伤的叶子或切碎的叶子浸在农杆菌溶液中，然后在合适的培养基中培养之。通过根癌农杆菌的植物转化由例如，
除了转化之后不得不再生为完整植株的体细胞，还可以转化植物分生组织的细胞，特别是可以发育成配子的那些细胞。在这种情况下，转化的配子循着天然植物的发育而产生转基因植物。因此，例如，用农杆菌处理拟南芥的种子，并从发育中的植物获得种子，其中一定比例的植物被转化因而是转基因的[Feldman，KA和Marks MD(1987).Mol Gen Genet208：274-289；Feldmann K(1992).在C Koncz，N-H Chua和J Shell编辑Methods in Arabidopsis Research.Word Scientific，Singapore，第274-289页]。可选的方法基于花序的反复去除以及莲座中心切割部位与转化农杆菌一起进行的孵育，由此在随后的时间点同样能够获得转化的种子(Chang(1994).Plant J.5：551-558；Katavic(1994).Mol Gen Genet，245：363-370)。然而，特别有效的方法是改良的真空浸润法，如“浸花法”(floral dip)。对于拟南芥的真空浸润，减压下用农杆菌悬液处理完整植株[Bechthold，N(1993).C R Acad Sci Paris Life Sci，316：1194-1199]，而对于“浸花法”，将发育中的花组织与表面活性剂处理的农杆菌悬液短暂孵育[Clough，SJ和Bent，AF(1998).The Plant J.16，735-743]。在两种情况下均收获一定比例的转基因种子，且可通过在上述选择性条件下培养而将这些种子与非转基因种子区分开来。另外，质体的稳定转化是有利的，因为质体在多数作物中为母系遗传，从而降低或消除了转基因通过花粉流失的风险。叶绿体基因组的转化通常通过Klaus等，2004[Nature Biotechnology 22(2)，225-229]系统展示的方法实现。简言之，将待转化的序列与可选择的标记基因一起克隆到同源于叶绿体基因组的侧翼序列之间。这些同源侧翼序列指导转基因位点特异性整合到质体基因组中。质体转化已在许多不同的植物物种中描述，且综述由Bock(2001)Transgenic plastids in basic research and plantbiotechnology.J Mol Biol.2001年9月21日；312(3)：425-38或Maliga，P(2003)Progress towards commercialization of plastid transformationtechnology.Trends Biotechnol.21，20-28给出。最近报道了其他生物技术进步，无标记的质体转化体，这可通过瞬时共整合的标记基因产生(Klaus等，2004，Nature Biotechnology 22(2)，225-229)。In addition to somatic cells which after transformation have to be regenerated into complete plants, it is also possible to transform cells of plant meristems, in particular those cells which can develop into gametes. In this case, the transformed gametes follow the development of the native plant to give rise to the transgenic plant. Thus, for example, the seeds of Arabidopsis thaliana are treated with Agrobacterium and seeds are obtained from developing plants in which a certain proportion of the plants are transformed and thus transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:274 -289; Feldmann K (1992). In C Koncz, N-H Chua and J Shell, eds. Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. An alternative method is based on the repeated removal of inflorescences and the incubation of the rosette central cut with transformed Agrobacterium, whereby transformed seeds can also be obtained at subsequent time points (Chang (1994). Plant J. 5: 551-558 ; Katavic (1994). Mol Gen Genet, 245:363-370). However, a particularly effective method is a modified vacuum infiltration method such as the "floral dip". For the vacuum infiltration of Arabidopsis thaliana, the whole plant is treated with the Agrobacterium suspension under reduced pressure [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], and for the "flower soaking method", Developing floral tissue was briefly incubated with a surfactant-treated Agrobacterium suspension [Clough, SJ and Bent, AF (1998). The Plant J. 16, 735-743]. A certain proportion of transgenic seeds were harvested in both cases, and these seeds could be distinguished from non-transgenic seeds by culturing under the above-mentioned selective conditions. In addition, stable transformation of plastids is advantageous because plastids are maternally inherited in most crops, thereby reducing or eliminating the risk of loss of the transgene through pollen. The transformation of the chloroplast genome is usually realized by the method shown systematically by Klaus et al., 2004 [Nature Biotechnology 22(2), 225-229]. Briefly, the sequence to be transformed is cloned between flanking sequences homologous to the chloroplast genome, together with a selectable marker gene. These homologous flanking sequences direct the site-specific integration of the transgene into the plastid genome. Plastid transformation has been described in many different plant species and reviewed by Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312(3):425-38 or Maliga, P(2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28 given. Another biotechnological advance was recently reported, marker-free plastid transformants, which can be generated by transiently co-integrated marker genes (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
T-DNA激活标记T-DNA activation marker
T-DNA激活标记(Hayashi等Science(1992)1350-1353)包括将T-DNA[通常含有启动子(也可以是翻译增强子或内含子)]插入在目的基因的基因组区或基因编码区上游或下游10kb处，从而在构型上使启动子能够指导靶基因的表达。通常破坏天然启动子对靶基因表达的调控，而使基因落入新引入的启动子的控制下。启动子一般包含于T-DNA中。此T-DNA可以例如通过农杆菌感染而随机插入植物基因组中，并导致所插入T-DNA附近的基因的表达被修饰。得到的转基因植物由于位于引入的启动子附近的基因的修饰表达而表现出显性表型。T-DNA Activation Marking (Hayashi et al. Science (1992) 1350-1353) involves inserting T-DNA [usually containing a promoter (also translational enhancer or intron)] into the genomic region or gene coding region of the gene of interest 10kb upstream or downstream, so that the promoter is configured to direct the expression of the target gene. Usually, the regulation of the expression of the target gene by the natural promoter is disrupted, leaving the gene under the control of the newly introduced promoter. The promoter is generally included in the T-DNA. This T-DNA can be inserted randomly into the plant genome, for example by Agrobacterium infection, and lead to modified expression of genes in the vicinity of the inserted T-DNA. The resulting transgenic plants exhibit a dominant phenotype due to the modified expression of genes located near the introduced promoter.
TILLINGTILLING
术语“TILLING”为“靶向诱导的基因组局部损伤”(Targeted InducedLocal Lesions In Genomes)的缩写，是一种用于生成和/或鉴定编码具有修饰的表达和/或活性的蛋白质的核酸的诱变技术。TILLING还允许选择携带此类突变变体的植物。这些突变变体可以在强度、位置或时间(例如，如果突变影响启动子的话)上呈现出修饰的表达。这些突变变体可以比其天然形式基因呈现更高的活性。TILLING将高密度诱变和高通量筛选方法结合在一起。TILLING一般遵循的步骤有：(a)EMS诱变(Redei GP和Koncz C，(1992)In Methods in Arabidopsis Research，Koncz C，Chua NH，Schell J编辑，新加坡，World Scientific Publishing Co，第16-82页；Feldmann等，(1994)In Meyerowitz EM，Somerville CR编辑，Arabidopsis.冷泉港实验室出版社，冷泉港，纽约，第137-172页；Lightner J和Caspar T，(1998)InJ Martinez-Zapater，J Salinas编辑，Methods on Molecular Biology，82卷Humana Press，Totowa，NJ，第91-104页)；(b)DNA制备和个体合并；(c)目的区域的PCR扩增；(d)变性和退火以形成杂双链体；(e)DHPLC，其中合并物中存在的杂双链体在色谱图上检测为额外的峰；(f)突变个体的鉴定；和(g)突变PCR产物的测序。TILLING的方法是本领域公知的(McCallum等(2002)Nat Biotechnol 18：455-457，由Stemple综述(2004)NatRev Genet 5(2)：145-50)。The term "TILLING" is an abbreviation for "Targeted Induced Local Lesions In Genomes", which is a mutagenesis used to generate and/or identify nucleic acids encoding proteins with modified expression and/or activity technology. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may exhibit modified expression in strength, location or timing (eg if the mutation affects a promoter). These mutant variants may exhibit higher activity of the gene than its native form. TILLING combines high-density mutagenesis and high-throughput screening methods. The steps generally followed by TILLING are: (a) EMS mutagenesis (Redei GP and Koncz C, (1992) In Methods in Arabidopsis Research, edited by Koncz C, Chua NH, Schell J, Singapore, World Scientific Publishing Co, pp. 16-82 pp.; Feldmann et al., (1994) In Meyerowitz EM, Somerville CR eds., Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 137-172; Lightner J and Caspar T, (1998) InJ Martinez-Zapater, Edited by J Salinas, Methods on Molecular Biology, Vol. 82 Humana Press, Totowa, NJ, pp. 91-104); (b) DNA preparation and individual pooling; (c) PCR amplification of the region of interest; (d) denaturation and annealing to form heteroduplexes; (e) DHPLC, where heteroduplexes present in the pool are detected as additional peaks on the chromatogram; (f) identification of mutant individuals; and (g) sequencing of mutant PCR products. Methods of TILLING are well known in the art (McCallum et al. (2002) Nat Biotechnol 18: 455-457, reviewed by Stemple (2004) Nat Rev Genet 5(2): 145-50).
同源重组homologous recombination
同源重组允许向基因组中的规定选定位置引入所选的核酸。同源重组是生物科学中常规用于低等生物体如酵母或剑叶藓(physcomitrella)的标准技术。在植物中进行同源重组的方法已经不仅在模式植物中描述(Offringa等(1990)EMBO J.9(10)：3077-84)，而且也在作物植物，如稻中描述(Terada等(2002)Nat Biotech 20(10)：1030-4；Iida和Terada(2004)Curr Opin Biotechnol 15(2)：132-8)，并且存在无论靶生物种类的通常可应用的方法(Miller等，Nature Biotechnol.25，778-785，2007)。Homologous recombination allows the introduction of selected nucleic acids at defined selected locations in the genome. Homologous recombination is a standard technique routinely used in the biological sciences in lower organisms such as yeast or physcomitrella. Methods for homologous recombination in plants have been described not only in model plants (Offringa et al. (1990) EMBO J.9(10):3077-84), but also in crop plants such as rice (Terada et al. (2002 ) Nat Biotech 20(10):1030-4; Iida and Terada (2004) Curr Opin Biotechnol 15(2):132-8), and there are generally applicable methods regardless of the target organism species (Miller et al., Nature Biotechnol. 25, 778-785, 2007).
产量Yield
术语“产量”通常表示具有经济价值的可测量产出，其一般是与规定的作物、面积和/或时期相关的。各植物部分基于其数量、大小和/或重量对产量直接做出贡献，或者实际产量是年作物每平方米的产量，用总产量(既包括收获的产量也包括估定的产量)除以种植的平方米来确定。术语植物的“产量”可能与该植物的营养性生物质(根和/或枝条生物质)、繁殖器官、和/或繁殖体(如种子)相关。The term "yield" generally refers to measurable output of economic value, generally related to a defined crop, area and/or time period. Each plant part directly contributes to yield based on its number, size and/or weight, or actual yield is the yield per square meter of the annual crop, divided by the total yield (both harvested and estimated) divided by the planted to determine the square meter. The term "yield" of a plant may relate to the plant's vegetative biomass (root and/or shoot biomass), reproductive organs, and/or propagules (eg, seeds).
早期活力early vitality
“早期活力”是指活跃健康充分均衡的生长(特别是在植物生长的早期阶段)，其可以因植物适应性(fitness)增强引起，例如，由于植物更好地适应其环境(即，优化能源资源的利用以及在枝条和根之间的分配)引起。具有早期活力的植物也显示出增加的幼苗存活和更佳的作物齐苗，这往往产生高均匀度的田地(作物以齐整的方式生长，即大多数植物基本上同时达到各发育阶段)，以及往往是更优更高的产量。因此，早期活力可以通过测量多种因素来确定，如千粒重、萌发率、出苗率、幼苗生长、幼苗高度、根长度、根和枝条生物量，等等。"Early vigor" refers to active, healthy, well-balanced growth (especially in the early stages of plant growth), which may result from increased plant fitness, for example, as a result of a plant better adapting to its environment (i.e., optimizing energy resource utilization and distribution between shoots and roots). Plants with early vigor also show increased seedling survival and better crop uniformity, which tend to produce fields of high uniformity (crops grow in a uniform fashion, i.e. most plants reach each developmental stage substantially at the same time), and Often better and higher yields. Therefore, early vigor can be determined by measuring various factors such as thousand-grain weight, germination rate, emergence rate, seedling growth, seedling height, root length, root and shoot biomass, etc.
增加/提高/增强increase / improve / enhance
术语“增加”、“提高”或“增强”可互换，且在本申请意义上表示与文中所定义的对照植物相比，产量和/或生长多出至少3％、4％、5％、6％、7％、8％、9％或10％，优选至少15％或20％，更优选25％、30％、35％或40％。The terms "increase", "increase" or "enhance" are interchangeable and in the sense of the present application mean yield and/or growth of at least 3%, 4%, 5%, more, compared to control plants as defined herein. 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40%.
种子产量seed yield
增加的种子产量自身可表现为如下一项或多项：a)种子生物量(种子总重量)的增加，这可以是基于单粒种子和/或每植株和/或每平方米的增加；b)每植株花数的增加；c)增加的(饱满)种子数；d)增加的种子饱满率(其表达为饱满种子数与种子总数的比率)；e)增加的收获指数，其表达为可收获部分如种子的产量除以总生物量的比率；f)增加的千粒重(TKW)；和g)增加的一级圆锥花序(primary panicles)数，这通过计数饱满种子数和它们的总重量外推得到。TKW增加可来自于种子大小和/或种子重量的增加，并且也可来自胚和/或胚乳大小的增加。Increased seed yield may manifest itself in one or more of the following: a) an increase in seed biomass (total seed weight), which may be based on a single seed and/or per plant and/or per square meter; b ) an increase in the number of flowers per plant; c) an increased number of (filled) seeds; d) an increased seed filling rate (expressed as the ratio of the number of filled seeds to the total number of seeds); e) an increased harvest index, expressed as the available Ratio of yield of harvested fractions such as seeds divided by total biomass; f) increased thousand-kernel weight (TKW); and g) increased number of primary panicles by counting the number of filled seeds and their total weight Push it. An increase in TKW may result from an increase in seed size and/or seed weight, and may also result from an increase in embryo and/or endosperm size.
种子产量的增加也可表现为种子大小和/或种子体积的增加。此外，种子产量的增加自身也可表现为种子面积和/或种子长度和/或种子宽度和/或种子周长的增加。增加的种子产量也可以导致改变的构造，或可以因改变的构造而发生。An increase in seed yield may also be manifested as an increase in seed size and/or seed volume. Furthermore, an increase in seed yield may also manifest itself as an increase in seed area and/or seed length and/or seed width and/or seed girth. Increased seed yield may also result in, or may occur as a result of, altered architecture.
绿度指数Greenness Index
如本文所用的“绿度指数”根据植物的数字图像计算。对于图像中属于植物目标的每一个像素，计算绿值相对于红值之比(在RGB模型中用于色度编码)。绿度指数表达为绿红比超过给定阈值的像素百分比。在正常生长条件下、在盐胁迫生长条件下、在养分可利用度下降的生长条件下，在开花前的末次成像中测量植物绿度指数。相反，在干旱胁迫生长条件下，在干旱后的首次成像中测量植物绿度指数。"Greenness Index" as used herein is calculated from digital images of plants. For each pixel in the image that belongs to a plant object, the ratio of the green value to the red value (used for chrominance encoding in the RGB model) is calculated. The greenness index is expressed as the percentage of pixels whose green-to-red ratio exceeds a given threshold. Plant greenness index was measured at the last imaging before flowering under normal growth conditions, under salt-stressed growth conditions, and under growth conditions with reduced nutrient availability. In contrast, under drought-stressed growth conditions, the plant greenness index was measured in the first imaging after drought.
植物plant
本文所用术语“植物”涵盖整株植物、植物的祖先和后代以及植物部分，包括种子、枝条、茎、叶、根(包括块茎)、花以及组织和器官，其中上述每一种都含有目的基因/核酸。术语“植物”也涵盖植物细胞、悬浮培养物、愈伤组织、胚、分生组织区、配子体、孢子体、花粉和小孢子，同样其中上述每一种都含有目的基因/核酸。The term "plant" as used herein encompasses whole plants, ancestors and descendants of plants, and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, each of which contains the gene of interest /nucleic acid. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, each of which also contains the gene/nucleic acid of interest.
尤其可用于本发明方法的植物包括属于植物界(Viridiplantae)超家族的所有植物，尤其是单子叶植物和双子叶植物，包括饲料或饲料豆科植物、观赏植物、粮食作物、乔木或灌木，选自包括如下的列表：槭树属物种(Acerspp.)、猕猴桃属物种(Actinidia spp.)、秋葵属物种(Abelmoschus spp.)、剑麻(Agave sisalana)、冰草属物种(Agropyron spp.)、匍茎剪股颖(Agrostisstolonifera)、葱芹属物种(Allium spp.)、苋属物种(Amaranthus spp.)、滨草(Ammophila arenaria)、凤梨(Ananas comosus)、番荔枝属物种(Annonaspp.)、芹菜(Apium graveolens)、落花生属物种(Arachis spp.)、木波罗属物种(Artocarpus spp.)、石刁柏(Asparagus officinalis)、燕麦属物种(Avenaspp.)(如燕麦(Avena sativa)、野燕麦(Avena fatua)、比赞燕麦(Avenabyzantina)、Avena fatua var.sativa、杂种燕麦(Avena hybrida))、阳桃(Averrhoa carambola)、簕竹属物种(Bambusa sp.)、冬瓜(Benincasahispida)、巴西栗(Bertholletia excelsea)、甜菜(Beta vulgaris)、芸苔属物种(Brassica spp.)(如欧洲油菜(Brassica napus)、甘蓝型油菜(Brassica rapassp.)[芸苔、油菜籽油菜、芜菁])、Cadaba farinosa、大叶茶(Camelliasinensis)、美人蕉(Canna indica)、大麻(Cannabis sativa)、辣椒属物种(Capsicum spp.)、苔草(Carex elata)、番木瓜(Carica papaya)、大果假虎刺(Carissa macrocarpa)、山核桃属物种(Carya spp.)、红花(Carthamustinctorius)、栗属物种(Castanea spp.)、Ceiba pentandra、苦苣(Cichoriumendivia)、樟属物种(Cinnamomum spp.)、西瓜(Citrullus lanatus)、柑橘属物种(Citrus spp.)、椰子属物种(Cocos spp.)、咖啡属物种(Coffea spp.)、芋(Colocasia esculenta)、可拉属物种(Cola spp.)、黄麻属物种(Corchorus sp.)、芫荽(Coriandrum sativum)、榛属物种(Corylus spp.)、山楂属物种(Crataegus spp.)、番红花(Crocus sativus)、南瓜属物种(Cucurbita spp.)、香瓜属物种(Cucumis spp.)、菜蓟属物种(Cynara spp.)、胡萝卜(Daucuscarota)、山马蟥属物种(Desmodium spp.)、龙眼(Dimocarpus longan)、薯蓣属物种(Dioscorea spp.)、柿树属物种(Diospyros spp.)、稗属物种(Echinochloa spp.)、油棕属(Elaeis)(如非洲油棕(Elaeis guineensis)、美洲油棕(Elaeis oleifera))、穇子(Eleusine coracana)、埃塞俄比亚画眉草(Eragrostis tef)、蔗茅属物种(Erianthus sp.)、枇杷(Eriobotrya japonica)、桉属物种(Eucalyptus sp)、红仔果(Eugenia uniflora)、荞麦属物种(Fagopyrum spp.)、山毛榉属物种(Fagus spp.)、苇状羊茅(Festucaarundinacea)、无花果(Ficus carica)、金桔属物种(Fortunella spp.)、草莓属物种(Fragaria spp.)、银杏(Ginkgo biloba)、大豆属物种(Glycine spp.)(如大豆(Glycine max)、黄豆(Soja hispida)或大豆(Soja max))、陆地棉(Gossypium hirsutum)、向日葵属物种(Helianthus spp.)(如向日葵(Helianthus annus))、萱草(Hemerocallis fulva)、木槿属物种(Hibiscus spp.)、大麦属物种(Hordeum spp.)(如大麦(Hordeum vulgare))、甘薯(Ipomoeabatatas)、核桃属物种(Juglans spp.)、莴苣(Lactuca sativa)、山黧豆属物种(Lathyrus spp.)、兵豆(Lens culinaris)、亚麻(Linum usitatissimum)、荔枝(Litchi chinensis)、百脉根属物种(Lotus spp.)、棱角丝瓜(Luffa acutangula)、羽扇豆属物种(Lupinus spp.)、地杨梅(Luzula sylvatica)、番茄属物种(Lycopersicon spp.)(如番茄(Lycopersicon esculentum、Lycopersiconlycopersicum、Lycopersicon pyriforme)、硬皮豆属物种(Macrotylomaspp.)、苹果属物种(Malus spp.)、西印度樱桃(Malpighia emarginata)、曼密苹果(Mammea americana)、芒果(Mangifera indica)、木薯属物种(Manihotspp.)、人心果(Manilkara zapota)、紫花苜蓿(Medicago sativa)、草木樨属物种(Melilotus spp.)、薄荷属物种(Mentha spp.)、芒(Miscanthus sinensis)、苦瓜属物种(Momordica spp.)、黑桑(Morus nigra)、芭蕉属物种(Musaspp.)、烟草属物种(Nicotiana spp.)、木犀榄属物种(Olea spp.)、仙人掌属物种(Opuntia spp.)、Ornithopus spp.、稻属物种(Oryza spp.)(如稻(Oryzasativa)，阔叶稻(Oryza latifolia))、黍糜(Panicum miliaceum)、柳枝稷(Panicum virgatum)、鸡蛋果(Passiflora edulis)、欧防风(Pastinaca sativa)、狼尾草属物种(Pennisetum sp.)、鳄梨属物种(Persea spp.)、香芹(Petroselinum crispum)、虉草(Phalaris arundinacea)、菜豆属物种(Phaseolus spp.)、梯牧草(Phleum pratense)、刺葵属物种(Phoenix spp.)、南方芦苇(Phragmites australis)、酸浆属物种(Physalis spp.)、松属物种(Pinus spp.)、阿月浑子(Pistacia vera)、豌豆属物种(Pisum spp.)、早熟禾属物种(Poa spp.)、杨属物种(Populus spp.)、牧豆树属物种(Prosopis spp.)、李属物种(Prunus spp.)、番石榴属物种(Psidium spp.)、石榴(Punicagranatum)、西洋梨(Pyrus communis)、栎属物种(Quercus spp.)、萝卜(Raphanus sativus)、波叶大黄(Rheum rhabarbarum)、茶藨子属物种(Ribesspp.)、蓖麻(Ricinus communis)、悬钩子属物种(Rubus spp.)、甘蔗属物种(Saccharum spp.)、柳属物种(Salix sp.)、接骨木属物种(Sambucus spp.)、黑麦(Secale cereale)、胡麻属物种(Sesamum spp.)、白芥属物种(Sinapis sp.)、茄属物种(Solanum spp.)(如马铃薯(Solanum tuberosum)、红茄(Solanumintegrifolium)或番柿(Solanum lycopersicum))、两色蜀黍(Sorghumbicolor)、菠菜属物种(Spinacia spp.)、蒲桃属物种(Syzygium spp.)、万寿菊属物种(Tagetes spp.)、酸豆(Tamarindus indica)、可可树(Theobromacacao)、车轴草属物种(Trifolium spp.)、鸭茅状磨擦禾(Tripsacumdactyloides)、Triticale sp.、小黑麦(Triticosecale rimpaui)、小麦属物种(Triticum spp.)(如小麦(Triticum aestivum)、硬粒小麦(Triticum durum)、圆锥小麦(Triticum turgidum)、Triticum hybernum、马卡小麦(Triticummacha)、面包小麦(Triticum sativum)、一粒小麦(Triticum monococcum)或普通小麦(Triticum vulgare))、小金莲花(Tropaeolum minus)、旱金莲(Tropaeolum majus)、越桔属物种(Vaccinium spp.)、野豌豆属物种(Viciaspp.)、豇豆属物种(Vigna spp.)、香堇菜(Viola odorata)、葡萄属物种(Vitisspp.)、玉蜀黍(Zea mays)、北美洲野生稻(Zizania palustris)、枣属物种(Ziziphus spp.)等等。Plants which are particularly useful in the method according to the invention include all plants belonging to the superfamily Viridiplantae, especially monocotyledonous and dicotyledonous plants, including forage or fodder legumes, ornamental plants, food crops, trees or shrubs, selected Self-inclusive list of: Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp. , Agrostisstolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annonas pp. , celery (Apium graveolens), Arachis spp., Artocarpus spp., Asparagus officinalis, Avenas pp. (such as oats (Avena sativa), Wild oats (Avena fatua), Bizan oats (Avenabyzantina), Avena fatua var. sativa, Hybrid oats (Avena hybrida)), Carambola (Averrhoa carambola), Bougainvillea sp. Brazil chestnut (Bertholletia excelsea), sugar beet (Beta vulgaris), Brassica spp. (such as Brassica napus), Brassica rapassp. [brassica, rapeseed rape, turnip] ), Cadaba farinosa, Camelliasinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Papaya Tiger thorn (Carissa macrocarpa), Hickory species (Carya spp.), safflower (Carthamustinctorius), chestnut species (Castanea spp.), Ceiba pentandra, chicory (Cichoriumendivia), camphor species (Cinnamo mum spp.), Watermelon (Citrullus lanatus), Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp. spp.), Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp.), Cucumis spp., Cynara spp., Carrot (Daucuscarota), Desmodium spp., Dimocarpus longan, Dioscorea spp.), Diospyros spp., Echinochloa spp., Elaeis (e.g. African oil palm (Elaeis guineensis), American oil palm (Elaeis oleifera)), barnyard (Eleusine coracana), Ethiopian teff (Eragrostis tef), Erianthus sp., loquat (Eriobotrya japonica), Eucalyptus sp., Eugenia uniflora, buckwheat (Fagopyrum spp.), Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba ), Glycine spp. (such as Glycine max, Soja hispida or soybean (Soja max)), upland cotton (Gossypium hirsutum), Helianthus spp. (such as sunflower ( Helianthus annus)), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (such as barley (Hordeum vulgare)), Sweet potato (Ipomoea batatas), Juglans spp., Lettuce (Lactuca sativa), Lathyrus spp., Lentil (Lens culinaris), Flax (Linum usitatissimum), Litchi (Litchi chinensis), Litchi Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (such as tomato (Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme), Macrotylomas pp., Malus spp., Acerola (Malpighia emarginata), Manmi apple (Mammea americana), Mango (Mangifera indica), Cassava (Manihotspp. .), Sapodilla (Manilkara zapota), Alfalfa (Medicago sativa), Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp. , Black Mulberry (Morus nigra), Musas pp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza Species (Oryza spp.) (such as rice (Oryza sativa), broad-leaved rice (Oryza latifolia)), millet (Panicum miliaceum), switchgrass (Panicum virgatum), egg fruit (Passiflora edulis), parsnip (Pastinaca sativa), wolf Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoebe spp. (Phoenix spp.), Southern Phragmites (Ph ragmites australis), Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp. , Populus spp., Prosopis spp., Prunus spp., Psidium spp., Pomegranate (Punicagranatum), Pear (Pyrus communis ), Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribess pp., Ricinus communis, Rubus spp. ), Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., White Mustard (Sinapis sp.), Solanum spp. (such as potato (Solanum tuberosum), tomato (Solanum integrifolium) or tomato (Solanum lycopersicum)), two-color sorghum (Sorghumbicolor), spinach (Spinacia spp. ), Syzygium spp., Tagetes spp., Tamarindus indica, Cocoa (Theobromacacao), Trifolium spp., Dactylis spp. (Tripsacumdactyloides), Triticale sp., Triticale (Triticosescale rimpaui), Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Maca wheat (Triticum macha, bread wheat (Triticum sativum), einkorn wheat (Triticum monococcum) or common wheat (Triticum vulgare)), nasturtium (Tropaeolum minus), Nasturtium (Tropaeolum majus), Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp. ( Vitis pp.), maize (Zea mays), North American wild rice (Zizania palustris), Ziziphus spp., etc.
发明详述Detailed description of the invention
现已令人惊讶地发现，在植物中调节编码alfin样多肽的核酸的表达可以产生相对于对照植物具有增强的非生物胁迫耐受性的植物。根据第一实施方案，本发明提供了相对于对照植物而增强植物的对多种非生物胁迫的耐受性的方法，包括调节编码alfin样多肽的核酸在植物中的表达和任选地选择具有增强的对非生物胁迫的耐受性的植物。It has now surprisingly been found that modulating expression in plants of a nucleic acid encoding an alfin-like polypeptide gives plants having increased tolerance to abiotic stress relative to control plants. According to a first embodiment, the present invention provides a method for enhancing the tolerance of a plant to a variety of abiotic stresses relative to control plants, comprising regulating expression in a plant of a nucleic acid encoding an alfin-like polypeptide and optionally selecting a plant with Plants with enhanced tolerance to abiotic stress.
此外，现已令人惊讶地发现，在植物中调节编码YRP多肽的核酸的表达可以产生相对于对照植物具有增强的非生物胁迫耐受性的植物。根据第一实施方案，本发明提供了相对于对照植物而增强植物的对多种非生物胁迫的耐受性的方法，包括调节编码YRP多肽的核酸在植物中的表达和任选地选择具有增强的对非生物胁迫的耐受性的植物。Furthermore, it has now surprisingly been found that modulating expression in plants of a nucleic acid encoding a YRP polypeptide can give plants having increased tolerance to abiotic stress relative to control plants. According to a first embodiment, the present invention provides a method for enhancing the tolerance of a plant to a variety of abiotic stresses relative to control plants, comprising regulating expression in a plant of a nucleic acid encoding a YRP polypeptide and optionally selecting a plant with enhanced Plants that are tolerant to abiotic stress.
此外，现已令人惊讶地发现，在植物中增强编码如本文中定义的BRXL多肽的核酸序列的表达可以产生相对于对照植物具有增强的产量相关性状的植物。根据第一实施方案，本发明提供了相对于对照植物而增强植物的产量相关性状的方法，包括增加编码BRXL多肽的核酸序列在植物中的表达。Furthermore, it has surprisingly been found that enhanced expression in plants of a nucleic acid sequence encoding a BRXL polypeptide as defined herein gives plants having enhanced yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a BRXL polypeptide.
本发明还提供了迄今未知的编码BRXL多肽的核酸序列和BRXL多肽。The present invention also provides hitherto unknown nucleic acid sequences encoding BRXL polypeptides and BRXL polypeptides.
根据本发明的一个实施方案，从而提供了分离的核酸分子，其选自：According to one embodiment of the invention, there is thus provided an isolated nucleic acid molecule selected from:
(i)如SEQ ID NOs：75、SEQ ID NOs：77或SEQ ID NOs：79的任一个所示的核酸序列；(i) a nucleic acid sequence as shown in any one of SEQ ID NOs: 75, SEQ ID NOs: 77 or SEQ ID NOs: 79;
(ii)如SEQ ID NOs：75、SEQ ID NOs：77或SEQ ID NOs：79的任一个所示的核酸序列的互补序列；(ii) as SEQ ID NOs:75, SEQ ID NOs:77 or the complementary sequence of any one of SEQ ID NOs:79 shown nucleic acid sequence;
(iii)编码BRXL多肽的核酸序列，所述多肽按照递增的优选次序与如SEQ ID NO：76、SEQ ID NO：78或SEQ ID NO：80的任意一个所示的多肽序列具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的氨基酸序列同一性。(iii) a nucleic acid sequence encoding a BRXL polypeptide, said polypeptide having at least 50% of the polypeptide sequence shown in any one of SEQ ID NO: 76, SEQ ID NO: 78 or SEQ ID NO: 80 in an increasing preferred order, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity.
根据本发明的另外的实施方案，也提供了分离的多肽，其选自：According to additional embodiments of the present invention, there is also provided an isolated polypeptide selected from the group consisting of:
(i)如SEQ ID NO：76、SEQ ID NO：78或SEQ ID NO：80的任一个所示的多肽序列；(i) a polypeptide sequence as shown in any one of SEQ ID NO:76, SEQ ID NO:78 or SEQ ID NO:80;
(ii)多肽序列，所述多肽序列按照递增的优选次序与如SEQ ID NO：76、SEQ ID NO：78或SEQ ID NO：80的任一个所示的多肽序列具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的氨基酸序列同一性；(ii) a polypeptide sequence having at least 50%, 55%, 55%, or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity;
(iii)上文(i)或(ii)所给出的任何多肽序列的衍生物。(iii) Derivatives of any of the polypeptide sequences given in (i) or (ii) above.
现已令人惊讶地发现，在植物中调节编码silky-1样多肽的核酸的表达可以产生相对于对照植物具有增强的非生物胁迫耐受性的植物。根据第一实施方案，本发明提供了相对于对照植物而增强植物对多种非生物胁迫的耐受性的方法，包括调节编码silky-1样多肽的核酸在植物中的表达和任选地选择具有增强的对非生物胁迫的耐受性的植物。It has now surprisingly been found that modulating expression in plants of a nucleic acid encoding a silky-1-like polypeptide gives plants having increased tolerance to abiotic stress relative to control plants. According to a first embodiment, the present invention provides a method for enhancing the tolerance of a plant to various abiotic stresses relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a silky-1-like polypeptide and optionally selecting Plants with enhanced tolerance to abiotic stress.
此外，现已令人惊讶地发现，在植物中调节编码如本文中定义的ARP6多肽的核酸的表达可以产生相对于对照植物具有增强的产量相关性状的植物。根据第一实施方案，本发明提供了相对于对照植物而增强植物的产量相关性状的方法，包括调节编码ARP6多肽的核酸在植物中的表达和任选地选择具有增强的产量相关性状的植物。Furthermore, it has surprisingly been found that modulating expression in plants of a nucleic acid encoding an ARP6 polypeptide as defined herein gives plants having enhanced yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding an ARP6 polypeptide and optionally selecting plants with enhanced yield-related traits.
此外，现已令人惊讶地发现，在植物中调节编码如本文中定义的脯氨酰寡肽酶(在下文中称为“POP多肽”)的核酸的表达可以产生相对于对照植物具有增强的产量相关性状的植物。根据第一实施方案，本发明提供了相对于对照植物而增强植物的产量相关性状的方法，包括调节编码POP多肽的核酸在植物中的表达和任选地选择具有增强的产量相关性状的植物。Furthermore, it has now surprisingly been found that modulating expression in plants of a nucleic acid encoding a prolyl oligopeptidase as defined herein (hereinafter referred to as "POP polypeptide") can result in enhanced yield relative to control plants plants with related traits. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a POP polypeptide and optionally selecting plants with enhanced yield-related traits.
此外，现已令人惊讶地发现，在植物中调节编码如本文中定义的CRL多肽的核酸的表达可以产生相对于对照植物具有增强的产量相关性状的植物。根据第一实施方案，本发明提供了相对于对照植物而增强植物的产量相关性状的方法，包括调节编码CRL多肽的核酸在植物中的表达和任选地选择具有增强的产量相关性状(优选种子总重量(totalwgseeds)、饱满种子数(nrfilledseed)、饱满率(fillrate)和收获指数(harvestindex)的任一个)的植物，和/或通过选择过表达CRL基因(引入的或内源性的)和/或CRL蛋白的转基因植物来鉴定该转基因植物。Furthermore, it has surprisingly been found that modulating expression in plants of a nucleic acid encoding a CRL polypeptide as defined herein gives plants having enhanced yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a CRL polypeptide and optionally selecting seeds with enhanced yield-related traits (preferably seeds total weight (totalwgseeds), number of filled seeds (nrfilledseed), filling rate (fillrate) and harvest index (harvestindex) any one of the plants, and/or by selection of overexpressed CRL genes (introduced or endogenous) and and/or CRL protein transgenic plants to identify the transgenic plants.
调节(优选，增加)编码alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸的表达的一个优选方法是，向植物中引入和表达编码alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸。A preferred method of modulating (preferably, increasing) the expression of a nucleic acid encoding an alfin-like polypeptide or a YRP polypeptide or a BRXL polypeptide or a silky-1-like polypeptide or an ARP6 polypeptide or a POP polypeptide or a CRL polypeptide is to introduce and express an alfin-like A nucleic acid of a polypeptide or a YRP polypeptide or a BRXL polypeptide or a silky-1-like polypeptide or an ARP6 polypeptide or a POP polypeptide or a CRL polypeptide.
涉及alfin样多肽，下文中对“用于本发明方法的蛋白质”的任何提及，均旨在指如本文中定义的alfin样多肽。下文中对“用于本发明方法的核酸”的任何提及，均旨在指能够编码这样的alfin样多肽的核酸。待引入植物中(并因此可以用于实施本发明方法)的该核酸是编码现将进行描述的此类蛋白质的任何核酸，在下文中也称为“alfin样核酸”或“alfin样基因”。Any reference hereinafter to "a protein useful in the methods of the invention" in reference to an alfin-like polypeptide is intended to refer to an alfin-like polypeptide as defined herein. Any reference hereinafter to a "nucleic acid useful in the methods of the invention" is intended to refer to a nucleic acid capable of encoding such an alfin-like polypeptide. The nucleic acid to be introduced into a plant (and thus can be used to carry out the methods of the invention) is any nucleic acid encoding a protein of the type now to be described, hereinafter also referred to as "alfin-like nucleic acid" or "alfin-like gene".
涉及YRP多肽，下文中对“用于本发明方法的蛋白质”的任何提及，均旨在指如本文中定义的YRP多肽。下文中对“用于本发明方法的核酸”的任何提及，均旨在指能够编码这样的YRP多肽的核酸。待引入植物中(并因此可以用于实施本发明方法)的该核酸是编码现将进行描述的此类蛋白质的任何核酸，在下文中也称为“YRP核酸”或“YRP基因”。Any reference hereinafter to a "protein useful in the methods of the invention" in reference to a YRP polypeptide is intended to refer to a YRP polypeptide as defined herein. Any reference hereinafter to a "nucleic acid useful in the methods of the invention" is intended to refer to a nucleic acid capable of encoding such a YRP polypeptide. The nucleic acid to be introduced into a plant (and thus can be used to carry out the methods of the invention) is any nucleic acid encoding a protein of the type now to be described, hereinafter also referred to as "YRP nucleic acid" or "YRP gene".
大麦(Hordeum vulgare)的YRP(SEQ ID NO：11和13)是编码GARP类转录因子成员的转录因子。YRP (SEQ ID NO: 11 and 13) of barley (Hordeum vulgare) is a transcription factor encoding a member of the GARP class of transcription factors.
涉及BRXL多肽，下文中对“用于本发明方法的蛋白质”的任何提及，均旨在指如本文中定义的BRXL多肽。下文中对“用于本发明方法的核酸序列”的任何提及，均旨在指能够编码这样的BRXL多肽的核酸序列。待引入植物中(并因此可以用于实施本发明方法)的该核酸序列是编码现将进行描述的此类蛋白质的任何核酸序列，在下文中也称为“BRXL核酸序列”或“BRXL基因”。Any reference hereinafter to "a protein useful in the methods of the invention" in reference to a BRXL polypeptide is intended to refer to the BRXL polypeptide as defined herein. Any reference hereinafter to a "nucleic acid sequence useful in the methods of the invention" is intended to refer to a nucleic acid sequence capable of encoding such a BRXL polypeptide. The nucleic acid sequence to be introduced into a plant (and thus can be used to carry out the method of the invention) is any nucleic acid sequence encoding a protein of the type which will now be described, hereinafter also referred to as "BRXL nucleic acid sequence" or "BRXL gene".
涉及silky-1样多肽，下文中对“用于本发明方法的蛋白质”的任何提及，均旨在指如本文中定义的silky-1样多肽。下文中对“用于本发明方法的核酸”的任何提及，均旨在指能够编码这样的silky-1样多肽的核酸。待引入植物中(并因此可以用于实施本发明方法)的该核酸是编码现将进行描述的此类蛋白质的任何核酸，在下文中也称为“silky-1样核酸”或“silky-1样基因”。Any reference hereinafter to "a protein useful in the method of the invention" in reference to a silky-1-like polypeptide is intended to refer to a silky-1-like polypeptide as defined herein. Any reference hereinafter to a "nucleic acid useful in the methods of the invention" is intended to refer to a nucleic acid capable of encoding such a silky-1-like polypeptide. The nucleic acid to be introduced into a plant (and thus can be used to carry out the methods of the invention) is any nucleic acid encoding a protein of the type which will now be described, hereinafter also referred to as "silky-1-like nucleic acid" or "silky-1-like Gene".
涉及ARP6多肽，下文中对“用于本发明方法的蛋白质”的任何提及，均旨在指如本文中定义的ARP6多肽。下文中对“用于本发明方法的核酸”的任何提及，均旨在指能够编码这样的ARP6多肽的核酸。待引入植物中(并因此可以用于实施本发明方法)的该核酸是编码现将进行描述的此类蛋白质的任何核酸，在下文中也称为“ARP6核酸”或“ARP6基因”。Any reference hereinafter to "a protein useful in the methods of the invention" in reference to an ARP6 polypeptide is intended to refer to the ARP6 polypeptide as defined herein. Any reference hereinafter to a "nucleic acid useful in the methods of the invention" is intended to refer to a nucleic acid capable of encoding such an ARP6 polypeptide. The nucleic acid to be introduced into a plant (and thus can be used to carry out the methods of the invention) is any nucleic acid encoding a protein of the type which will now be described, hereinafter also referred to as "ARP6 nucleic acid" or "ARP6 gene".
涉及POP多肽，下文中对“用于本发明方法的蛋白质”的任何提及，均旨在指如本文中定义的POP多肽。下文中对“用于本发明方法的核酸”的任何提及，均旨在指能够编码这样的POP多肽的核酸。待引入植物中(并因此可以用于实施本发明方法)的该核酸是编码现将进行描述的此类蛋白质的任何核酸，在下文中也称为“POP核酸”或“POP基因”。Any reference hereinafter to "a protein useful in the methods of the invention" in reference to a POP polypeptide is intended to refer to a POP polypeptide as defined herein. Any reference hereinafter to a "nucleic acid useful in the methods of the invention" is intended to refer to a nucleic acid capable of encoding such a POP polypeptide. The nucleic acid to be introduced into a plant (and thus can be used to carry out the methods of the invention) is any nucleic acid encoding a protein of the type now to be described, hereinafter also referred to as "POP nucleic acid" or "POP gene".
涉及CRL多肽，下文中对“用于本发明方法的蛋白质”的任何提及，均旨在指如本文中定义的CRL多肽。下文中对“用于本发明方法的核酸”的任何提及，均旨在指能够编码这样的CRL多肽的核酸。待引入植物中(并因此可以用于实施本发明方法)的该核酸是编码现将进行描述的此类蛋白质的任何核酸，在下文中也称为“CRL核酸”或“CRL基因”。Any reference hereinafter to "a protein useful in the methods of the invention" in reference to a CRL polypeptide is intended to refer to a CRL polypeptide as defined herein. Any reference hereinafter to a "nucleic acid useful in the methods of the invention" is intended to refer to a nucleic acid capable of encoding such a CRL polypeptide. The nucleic acid to be introduced into a plant (and thus can be used to carry out the methods of the invention) is any nucleic acid encoding a protein of the type now to be described, hereinafter also referred to as "CRL nucleic acid" or "CRL gene".
本文中定义的“alfin样多肽”是指包含GNGGTG或GTGGNG的核心六聚体基序的任何多肽。An "alfin-like polypeptide" as defined herein refers to any polypeptide comprising the core hexamer motif of GNGGTG or GTGGNG.
此类alfin样多肽的实例包括SEQ ID NO：2和SEQ ID NO：4的任一个所示的序列的直向同源物和旁系同源物。Examples of such alfin-like polypeptides include orthologues and paralogues of the sequences shown in any one of SEQ ID NO: 2 and SEQ ID NO: 4.
Alfin样多肽和其直向同源物和旁系同源物通常按照递增的优选次序与SEQ ID NO：2和SEQ ID NO：4的任一个所示的氨基酸序列具有至少25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性。Alfin-like polypeptides and their orthologs and paralogs generally have at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% , 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60 %, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% overall sequence identity.
使用全局比对算法，例如程序GAP(GCG Wisconsin Package，Accelrys)中的Needleman Wunsch算法，优选利用缺省参数和优选利用成熟蛋白质的序列(即，不考虑分泌信号或转运肽)，确定全序列同一性。与全序列同一性相比，当只考虑保守结构域或基序时，序列同一性通常更高。Full sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with the sequence of the mature protein (i.e., regardless of secretion signals or transit peptides) sex. Sequence identity is usually higher when only conserved domains or motifs are considered compared to full sequence identity.
优选，所述多肽序列，当其用于系统发生树的构建时，与包含SEQ IDNO：2和SEQ ID NO：4所示的氨基酸序列的alfin样多肽组而非与任何其他组聚类。用于系统发生树的构建和分析的工具和技术在本领域内是公知的。Preferably, said polypeptide sequence, when used in the construction of a phylogenetic tree, clusters with the group of alfin-like polypeptides comprising the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 rather than with any other group. Tools and techniques for construction and analysis of phylogenetic trees are well known in the art.
如本文中所定义的“YRP多肽”是指根据SEQ ID NO：11和SEQ IDNO：13的任何多肽、和SEQ ID NO：11和SEQ ID NO：13的任一个所示的序列的直向同源物和旁系同源物。"YRP polypeptide" as defined herein refers to any polypeptide according to SEQ ID NO: 11 and SEQ ID NO: 13, and the orthoisomorphic sequence of any one of SEQ ID NO: 11 and SEQ ID NO: 13 Paralogues and paralogues.
YRP多肽和其直向同源物和旁系同源物通常按照递增的优选次序与SEQ ID NO：11和SEQ ID NO：13的任一个所示的氨基酸序列具有至少25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、5l％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性。YRP polypeptides and their orthologs and paralogues generally have at least 25%, 26%, 27% of the amino acid sequence shown in any one of SEQ ID NO: 11 and SEQ ID NO: 13 in an increasing preferred order. %, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 5l%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% , 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77 %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% overall sequence identity.
使用全局比对算法，例如程序GAP(GCG Wisconsin Package，Accelrys)中的Needleman Wunsch算法，优选利用缺省参数和优选利用成熟蛋白质的序列(即，不考虑分泌信号或转运肽)，确定全序列同一性。与全序列同一性相比，当只考虑保守结构域或基序时，序列同一性通常更高。Full sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with the sequence of the mature protein (i.e., regardless of secretion signals or transit peptides) sex. Sequence identity is usually higher when only conserved domains or motifs are considered compared to full sequence identity.
优选，所述多肽序列，当其用于系统发生树的构建时，与包含SEQ IDNO：11和SEQ ID NO：13所示的氨基酸序列的YRP多肽组而非与任何其他组聚类。用于系统发生树的构建和分析的工具和技术在本领域内是公知的。Preferably, said polypeptide sequence, when it is used in the construction of a phylogenetic tree, is clustered with the YRP polypeptide group comprising the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 13 rather than with any other group. Tools and techniques for construction and analysis of phylogenetic trees are well known in the art.
如本文中所定义的“BRXL多肽”是指包含具有InterPro entryIPR013591 DZC结构域(PFAM entry PF08381 DZC)的至少两个BRX结构域的任意多肽。A "BRXL polypeptide" as defined herein refers to any polypeptide comprising at least two BRX domains having an InterPro entry IPR013591 DZC domain (PFAM entry PF08381 DZC).
可选地或此外地，本文中定义的“BRXL多肽”是指任何这样的多肽，其包含：(i)按照递增的优选次序与SEQ ID NO：81所示的BRX结构域至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高氨基酸序列同一性；和(ii)按照递增的优选次序与SEQ ID NO：82所示的BRX结构域至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高氨基酸序列同一性。Alternatively or additionally, a "BRXL polypeptide" as defined herein refers to any polypeptide comprising: (i) at least 50%, 55% of the BRX domain shown in SEQ ID NO: 81 in an increasing order of preference %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher amino acid sequence identity; and (ii) in increasing order of preference with SEQ ID NO: 82 at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acids in the BRX domain sequence identity.
可选地或此外地，本文中定义的“BRXL多肽”是指任何这样的多肽，其包含：(i)按照递增的优选次序与SEQ ID NO：83所示的保守结构域1(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性；和(ii)按照递增的优选次序与SEQ ID NO：84所示的保守结构域2(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。Alternatively or additionally, "BRXL polypeptide" as defined herein refers to any such polypeptide, which comprises: (i) the conserved
此外，本文中定义的“BRXL多肽”可以还包含：(i)按照递增的优选次序与SEQ ID NO：85所示的保守结构域3至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高氨基酸序列同一性；和(ii)按照递增的优选次序与SEQ ID NO：86所示的保守结构域4至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高氨基酸序列同一性。In addition, the "BRXL polypeptide" defined herein may further comprise: (i) at least 50%, 55%, 60%, 65%, and 70% of the conserved domain 3 shown in SEQ ID NO: 85 in an ascending preferred order , 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher amino acid sequence identity; and (ii) with the conserved domain shown in SEQ ID NO: 86 according to increasing
可选地或此外，本文中定义的“BRXL多肽”是指任何这样的多肽序列，其按照递增的优选次序与SEQ ID NO：18所示的多肽具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高氨基酸序列同一性。Alternatively or in addition, "BRXL polypeptide" as defined herein refers to any polypeptide sequence that has at least 40%, 45%, 50%, 55% of the polypeptide shown in SEQ ID NO: 18 in an increasing preferred order. %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity.
可选地或此外，本文中定义的“BRXL多肽”是指任何这样的多肽，其按照递增的优选次序与本文中表A3给出的任一多肽序列具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高氨基酸序列同一性。Alternatively or in addition, a "BRXL polypeptide" as defined herein refers to any polypeptide which, in increasing order of preference, has at least 40%, 45%, 50% of any polypeptide sequence given in Table A3 herein , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher amino acid sequence identity.
可选地或此外，本文中定义的“BRXL多肽”是指任何这样的多肽，其在酵母双杂交测定中与其本身或与另一个BRLX多肽相互作用。Alternatively or additionally, a "BRXL polypeptide" as defined herein refers to any polypeptide that interacts with itself or with another BRLX polypeptide in a yeast two-hybrid assay.
如本文中所定义的“silky-1样多肽”是指由SEQ ID NO：91、SEQ IDNO：93和SEQ ID NO：95的任一个所表示的任何多肽以及其直向同源物和旁系同源物。A "silky-1-like polypeptide" as defined herein refers to any polypeptide represented by any one of SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 95 and its orthologs and paralogs Homologs.
Silky-1样多肽和其直向同源物和旁系同源物通常按照递增的优选次序与SEQ ID NO：91、SEQ ID NO：93和SEQ ID NO：95的任一个所示的氨基酸具有至少25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性。Silky-1-like polypeptides and their orthologues and paralogues generally have amino acids shown in any one of SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 95 in an increasing preferred order. At least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% , 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity.
使用全局比对算法，例如程序GAP(GCG Wisconsin Package，Accelrys)中的Needleman Wunsch算法，优选利用缺省参数和优选利用成熟蛋白质的序列(即，不考虑分泌信号或转运肽)，确定全序列同一性。与全序列同一性相比，当只考虑保守结构域或基序时，序列同一性通常更高。Full sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with the sequence of the mature protein (i.e., regardless of secretion signals or transit peptides) sex. Sequence identity is usually higher when only conserved domains or motifs are considered compared to full sequence identity.
优选，所述多肽序列，当其用于系统发生树的构建时，与包含SEQ IDNO：91、SEQ ID NO：93和SEQ ID NO：95所示的氨基酸序列的silky-1样多肽组而非与任何其他组聚类。用于系统发生树的构建和分析的工具和技术在本领域内是公知的。Preferably, the polypeptide sequence, when used in the construction of a phylogenetic tree, is not related to the silky-1-like polypeptide group comprising the amino acid sequences shown in SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 95 Cluster with any other group. Tools and techniques for construction and analysis of phylogenetic trees are well known in the art.
如本文中所定义的ARP6多肽是指任何这样的多肽，其包含肌动蛋白结构域并按照递增的优选次序与SEQ ID NO：102或表A5的任何多肽所示的氨基酸具有至少30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性。An ARP6 polypeptide as defined herein refers to any polypeptide comprising an actin domain and having at least 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% overall sequence identity.
ARP6多肽通常包含肌动蛋白结构域。肌动蛋白结构域在本领域内是公知的。例如，Pfam蛋白质结构域数据库(Bateman等，Nucleic AcidsResearch 30(1)：276-280(2002))提及肌动蛋结构域为具有登录号：PF00022。ARP6 polypeptides typically comprise an actin domain. Actin domains are well known in the art. For example, the Pfam protein domain database (Bateman et al., Nucleic Acids Research 30(1):276-280 (2002)) refers to the actin domain as having accession number: PF00022.
Pfam PF00022结构域基于HMMER2包提供的隐马尔可夫模型(HMM)检索。在HMMER2中，如BLAST，计算E-值(期望值)。E-值是预期仅因偶然而具有相等或更好的分值的命中次数。良好的E值比1小得多。大约1是刚好预期因偶然而发生。原则上，为了决定匹配的显著性，仅需要E值。下列是定义Pfam数据库中提供的肌动蛋白结构域的结构域分值。Pfam PF00022 domains were retrieved based on the Hidden Markov Model (HMM) provided by the HMMER2 package. In HMMER2, like BLAST, E-value (expectation value) is calculated. The E-value is the number of hits expected to have an equal or better score simply by chance. A good E value is much smaller than 1. About 1 is just expected to happen by chance. In principle, to decide the significance of a match, only the E value is needed. The following are domain scores defining the actin domains provided in the Pfam database.
显示用于建立肌动蛋白结构域的HMM模型。将ls(全局)和fs(片段)匹配物与模型比对以产生全比对(full alignment)的顺序。该建立方法可以首先是全局的(其中首先比对ls匹配物，然后进行不重叠的fs匹配物的比对)、按分值的(其中按照E值分值的顺序，对匹配物进行比对)，或者可以首先是局部的(其中首先比对fs匹配物，然后进行不重叠的ls匹配物的比对)。显示与HMM比对的单结构域的分值。如果存在1个以上结构域，那么，对于该Pfam entry，序列分值是全部这些结构域分值的总和。如果只有一个结构域，那么该蛋白质的序列和结构域分值将是相同的。The HMM model used to build the actin domain is shown. The ls (global) and fs (fragment) hits are aligned to the model to generate a full alignment sequence. The establishment method can first be global (where ls matches are aligned first, followed by non-overlapping fs matches), scorewise (where matches are aligned in order of E-value scores ), or can be local first (where the fs matches are aligned first, followed by non-overlapping ls matches). Scores for single domains aligned to HMM are shown. If there is more than 1 domain, then, for this Pfam entry, the sequence score is the sum of the scores of all these domains. If there is only one domain, then the protein's sequence and domain scores will be the same.
显示肌动蛋白结构域的所使用的gathering截断值。该值是用于建立全比对的搜索阈值。Gathering截断值是为适于Pfam entry的全比对，一个序列必须达到的最小分值。对于每一个Pfam HMM，存在两个截断值——序列截断值和结构域截断值。The gathering cutoff used for the actin domain is shown. This value is the search threshold used to build full alignments. The Gathering cut-off value is the minimum score that a sequence must achieve in order to be suitable for the full alignment of Pfam entry. For each Pfam HMM, there are two cutoffs - a sequence cutoff and a domain cutoff.
Trusted截断值是指全比对中最低得分匹配的比特分值(bit score)。The Trusted cutoff value refers to the bit score of the lowest scoring match in the full alignment.
Noise截断值(NC)是指不在全比对内的最高得分匹配的比特分值。The Noise Cutoff (NC) is the bit score of the highest scoring match that is not in the full alignment.
可选地，ARP6蛋白的同源物按照递增的优选次序与SEQ ID NO：102所示的氨基酸具有至少25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性，条件是同源蛋白质包含上文所述的保守基序。可以使用全局比对算法，例如程序GAP(GCG Wisconsin Package，Accelrys)中的Needleman Wunsch算法，优选利用缺省参数和优选利用成熟蛋白质的序列(即，不考虑分泌信号或转运肽)，确定全序列同一性。与全序列同一性相比，当只考虑保守结构域或基序时，序列同一性通常更高。Alternatively, the homologue of the ARP6 protein has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32% of the amino acids shown in SEQ ID NO: 102 according to the increasing preferred order , 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49 %, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % overall sequence identity, provided that the homologous proteins contain the conserved motifs described above. The full sequence can be determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with the sequence of the mature protein (i.e., regardless of secretion signals or transit peptides) identity. Sequence identity is usually higher when only conserved domains or motifs are considered compared to full sequence identity.
优选，所述多肽序列，当其用于系统发生树例如Kandasamy等2004.Trends Plant Sci 9：196-202的图1中描述的系统发生树的构建时，与包含SEQ ID NO：102所示的AtARP6的氨基酸序列的ARP6多肽组而非与任何其他组聚类。Preferably, the polypeptide sequence, when it is used for the construction of a phylogenetic tree such as the phylogenetic tree described in Figure 1 of Kandasamy et al. 2004. Trends Plant Sci 9: 196-202, contains the sequence shown in SEQ ID NO: 102. The ARP6 polypeptide group of the amino acid sequence of AtARP6 does not cluster with any other group.
如本文中所定义的“脯氨酰寡肽酶”或“POP多肽是指在MEROPS数据库中分类为S9家族肽酶的任意丝氨酸蛋白酶。在该丝氨酸蛋白酶内，POP蛋白属于SC族(SC clan)中的α/β水解酶折叠(Tripathi&Sowdhamini，BMC Genomics，7，200，2006)。优选，POP多肽属于脯氨酰寡肽酶的S9B亚家族。可以用于本发明方法的POP多肽包括肽酶_S9结构域(Pfam登录号PF00326)和优选还包括DPPIV_N结构域(Pfam登录号PF00930)。"Prolyl oligopeptidase" or "POP polypeptide" as defined herein refers to any serine protease classified as S9 family peptidases in the MEROPS database. Within this serine protease, the POP protein belongs to the SC clan (SC clan) α/β hydrolase folding (Tripathi & Sowdhamini, BMC Genomics, 7,200,2006) in. Preferably, POP polypeptide belongs to the S9B subfamily of prolyl oligopeptidase. The POP polypeptide that can be used for the inventive method comprises peptidase- The S9 domain (Pfam accession number PF00326) and preferably also includes the DPPIV_N domain (Pfam accession number PF00930).
优选，POP多肽还包括下列基序的一个或多个：Preferably, the POP polypeptide further comprises one or more of the following motifs:
基序1(SEQ ID NO：118)：Motif 1 (SEQ ID NO: 118):
(V/S/L)(Y/H)GGP(V/S/L)(Y/H)GGP
基序2(SEQ ID NO：119)：Motif 2 (SEQ ID NO: 119):
(Q/A)(Y/F)(L/W)(R/T/S)(S/N)(R/Q/K/I)G(I/W/Y)(L/A/S)(V/F/Q)(W/V/A/L)(K/D/I)(L/M/V/I)(D/N)(N/Y)(R/G)G(S/T)(A/S/T/L)(R/G)(R/Y)G(L/R/E)(Q/A)(Y/F)(L/W)(R/T/S)(S/N)(R/Q/K/I)G(I/W/Y)(L/A/S )(V/F/Q)(W/V/A/L)(K/D/I)(L/M/V/I)(D/N)(N/Y)(R/G)G( S/T)(A/S/T/L)(R/G)(R/Y)G(L/R/E)
基序3(SEQ ID NO：120)：Motif 3 (SEQ ID NO: 120):
(R/H)(I/L)(G/C/T)(I/L/V)(Y/C/S/T/L)G(W/G/R)S(Y/A/H)GG(Y/F)(M/L/T)(A/S/T)(R/H)(I/L)(G/C/T)(I/L/V)(Y/C/S/T/L)G(W/G/R)S(Y/A/H )GG(Y/F)(M/L/T)(A/S/T)
基序4(SEQ ID NO：121)：Motif 4 (SEQ ID NO: 121):
(Y/F)(D/E)(T/S/A)(Y/H/F/R)(Y/G)(T/I/D)(E/D/Q)(K/N/S)(Y/L/H)(M/V/Y)(G/T)(Y/F)(D/E)(T/S/A)(Y/H/F/R)(Y/G)(T/I/D)(E/D/Q)(K/N/ S)(Y/L/H)(M/V/Y)(G/T)
基序5(SEQ ID NO：122)：Motif 5 (SEQ ID NO: 122):
S(V/I/P)(M/I)(H/N/S)(H/F)(V/I)S(V/I/P)(M/I)(H/N/S)(H/F)(V/I)
基序6(SEQ ID NO：123)：Motif 6 (SEQ ID NO: 123):
(H/Q/L)G(M/L/T)(I/E/K)D(E/K/L)(N/V/R)V(H/T/P)(F/P/I)(H/Q/L)G(M/L/T)(I/E/K)D(E/K/L)(N/V/R)V(H/T/P)(F/P/ I)
基序7(SEQ ID NO：124)：Motif 7 (SEQ ID NO: 124):
(F/Y)(P/E)(D/G/N)(E/D)(R/Q/N)H(M/G/P)(P/F/L)(R/D)(G/R/K)。(F/Y)(P/E)(D/G/N)(E/D)(R/Q/N)H(M/G/P)(P/F/L)(R/D)( G/R/K).
更优选，POP多肽还包括下列基序的一个或多个：More preferably, the POP polypeptide further comprises one or more of the following motifs:
基序8(SEQ ID NO：125)：Motif 8 (SEQ ID NO: 125):
KLRRERLR(Q/E)RGLGVT(C/R)YEWKLRRERLR(Q/E)RGLGVT(C/R)YEW
基序9(SEQ ID NO：126)：Motif 9 (SEQ ID NO: 126):
HG(L/I)AEYIAQEEM(D/E)R(K/R)(N/T/M)G(Y/F)WWS(L/P)DSHG(L/I)AEYIAQEEM(D/E)R(K/R)(N/T/M)G(Y/F)WWS(L/P)DS
基序10(SEQ ID NO：127)：Motif 10 (SEQ ID NO: 127):
GFIWASE(K/R)(S/T)GFRHLGFIWASE(K/R)(S/T)GFRHL
基序11(SEQ ID NO：128)：Motif 11 (SEQ ID NO: 128):
LR(S/N)(Q/K/R)GILVWK(L/M)DLR(S/N)(Q/K/R)GILVWK(L/M)D
基序12(SEQ ID NO：129)：Motif 12 (SEQ ID NO: 129):
IG(L/V/I)(C/Y)GW SYGG(Y/F)IG(L/V/I)(C/Y)GW SYGG(Y/F)
基序13(SEQ ID NO：130)：Motif 13 (SEQ ID NO: 130):
CAV(S/A)GAPVT(S/A)WDGYDCAV(S/A)GAPVT(S/A)WDGYD
基序14(SEQ ID NO：131)：Motif 14 (SEQ ID NO: 131):
HGMIDENVHFRHTARLHGMIDENVHFRHTARL
更优选，POP多肽按照递增的优选次序包含至少2个、至少3个、至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个、至少11个或全部12个基序。More preferably, the POP polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 in increasing order of preference or all 12 motifs.
可选地，POP蛋白的同源物按照递增的优选次序与SEQ ID NO：117所示的氨基酸具有至少25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性，条件是同源蛋白质包含上文所述的保守基序中的至少一个。可以使用全局比对算法，例如程序GAP(GCG WisconsinPackage，Accelrys)中的Needleman Wunsch算法，优选利用缺省参数和优选利用成熟蛋白质的序列(即，不考虑分泌信号或转运肽)，确定全序列同一性。与全序列同一性相比，当只考虑保守结构域或基序时，序列同一性通常更高。优选，POP多肽中的基序按照递增的优选次序与SEQ ID NO：118至SEQ ID NO：129所示的基序(基序1至12)具有至少70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性。Alternatively, the homologue of the POP protein has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32% of the amino acids shown in SEQ ID NO: 117 in an increasing preferred order , 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49 %, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % overall sequence identity, provided that the homologous protein comprises at least one of the conserved motifs described above. Full sequence identity can be determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably using default parameters and preferably using the sequence of the mature protein (i.e., regardless of secretion signals or transit peptides) sex. Sequence identity is usually higher when only conserved domains or motifs are considered compared to full sequence identity. Preferably, the motifs in the POP polypeptide share at least 70%, 71%, 72%, 73% with the motifs (
优选，所述多肽序列，当其用于系统发生树例如图13(Tripathi&Sowdhamini 2006)中描述的系统发生树的构建时，与包含SEQ ID NO：117所示的氨基酸序列的箭头所指POP多肽组而非与任何其他组聚类。Preferably, said polypeptide sequence, when it is used for the construction of a phylogenetic tree such as the phylogenetic tree described in Figure 13 (Tripathi & Sowdhamini 2006), is the same as the POP polypeptide group indicated by the arrow comprising the amino acid sequence shown in SEQ ID NO: 117 rather than clustering with any other group.
如本文中定义的“CRL多肽”是指包含蛋白质结构域的任意多肽，所述蛋白质结构域按照递增的优选次序与SEQ ID NO：156的氨基酸42-236的序列所表示的氨基酸结构域具有至少45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性，并且所述多肽任选地具有跨膜结构域，优选如由SEQ ID NO：SEQ ID NO：156的氨基酸19-36的序列所表示的结构域。A "CRL polypeptide" as defined herein refers to any polypeptide comprising a protein domain having, in increasing order of preference, at least 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61% , 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% overall sequence identity, and the polypeptide optionally has a transmembrane domain, preferably as defined by SEQ ID NO: amino acids 19-36 of SEQ ID NO: 156 The domain represented by the sequence of .
确定跨膜结构域存在于蛋白质中的方法在本领域内是公知的(更详细的内容提供于实施例部分)。具有跨膜结构域的蛋白质预期定位于膜结构处。优选，本发明的蛋白质，当存在于细胞中时，定位在膜上。Methods for determining the presence of transmembrane domains in proteins are well known in the art (more details are provided in the Examples section). Proteins with transmembrane domains are expected to localize at membrane structures. Preferably, the protein of the invention, when present in a cell, is localized on the membrane.
本发明的优选蛋白质是指按照递增的优选次序与下列基序的任意一个或多个具有至少50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性的CRL多肽：The preferred protein of the present invention refers to at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% of any one or more of the following motifs in increasing order of preference , 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, CRL polypeptides with 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity:
(i)基序1：EQAFWRxPXKPFRQR(SEQ ID NO：207)；(i) Motif 1: EQAFWRxPXKPFRQR (SEQ ID NO: 207);
(ii)基序2：NFCDR(SEQ ID NO：208)；(ii) Motif 2: NFCDR (SEQ ID NO: 208);
(iii)基序3：RGKRCLYEGS(SEQ ID NO：209)；(iii) Motif 3: RGKRCLYEGS (SEQ ID NO: 209);
(iv)基序4：QVWGxKXGPYEFK(SEQ ID NO：210)；(iv) Motif 4: QVWGxKXGPYEFK (SEQ ID NO: 210);
其中X表示任意氨基酸。wherein X represents any amino acid.
可选地，CRL蛋白的同源物按照递增的优选次序与表A7中所示的任意氨基酸，优选与SEQ ID NO：156所示的序列具有至少25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、5l％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性。可以使用全局比对算法，例如程序GAP(GCG Wisconsin Package，Accelrys)中的Needleman Wunsch算法，优选利用缺省参数和优选利用成熟蛋白质的序列(即，不考虑分泌信号或转运肽)，确定全序列同一性。与全序列同一性相比，当只考虑保守结构域或基序时，序列同一性通常更高。对于局部比对，Smith-Waterman算法是特别有用的(Smith TF，Waterman MS(1981)J.Mol.Biol 147(1)；195-7)。确定序列同一性的一个备选方法由称为MATGAT(实施例部分)的方法提供。Optionally, the homologue of the CRL protein has at least 25%, 26%, 27%, 28%, any amino acid shown in Table A7, preferably the sequence shown in SEQ ID NO: 156 according to an increasing preferred order. 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% , 46%, 47%, 48%, 49%, 50%, 5l%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62 %, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% overall sequence identity. The full sequence can be determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with the sequence of the mature protein (i.e., regardless of secretion signals or transit peptides) identity. Sequence identity is usually higher when only conserved domains or motifs are considered compared to full sequence identity. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7). An alternative method of determining sequence identity is provided by the method known as MATGAT (Examples section).
优选，所述多肽序列，当其用于系统发生树例如图16中描述的系统发生树的构建时，与包含单子叶植物编码的氨基酸序列，更优选双子叶植物编码的序列，最优选SEQ ID NO：156所示序列，的CRL多肽组而非与任何其他组聚类。Preferably, said polypeptide sequence, when it is used for the construction of a phylogenetic tree such as the phylogenetic tree described in Fig. The sequence shown in NO: 156, the CRL polypeptide group is not clustered with any other group.
术语“结构域”、“标签序列”和“基序”在本文“定义”部分定义。存在用于鉴定结构域的专家数据库，例如SMART(Schultz等(1998)Proc.Natl.Acad.Sci.USA 95，5857-5864；Letunic等(2002)Nucleic Acids Res 30，242-244)、InterPro(Mulder等，(2003)Nucl.Acids.Res.31，315-318)、Prosite(Bucher和Bairoch(1994)，A generalized profile syntax forbiomolecular sequences motifs and its function in automatic sequenceinterpretation.(In)ISMB-94；第二届分子生物学智能系统国际会议记录(Proceedings 2nd International Conference on Intelligent Systems forMolecular Biology)Altman R.，Brutlag D.，Karp P.，Lathrop R.，Searls D.编辑，53-61页，AAAIPress，Menlo Park；Hulo等，Nucl.Acids.Res.32：D134-D137，(2004))或者Pfam(Bateman等，Nucleic Acids Research 30(1)：276-280(2002))。进行蛋白质序列芯片(in silico)分析的一组工具可以从ExPASy蛋白质组学服务器获得(瑞士生物信息学研究所(Swiss Institute ofBioinformatics)(Gasteiger等ExPASy：the proteomics server for in-depthprotein knowledge and analysis.Nucleic Acids Res 31：3784-3788(2003))。结构域或基序也可以利用常规技术例如通过序列比对来鉴定。The terms "domain", "tag sequence" and "motif" are defined herein in the "Definitions" section. There are expert databases for identifying domains such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro ( Mulder et al., (2003) Nucl.Acids.Res.31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings of the 2nd International Conference on Intelligent Systems for Molecular Biology (Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology) edited by Altman R., Brutlag D., Karp P., Lathrop R., Searls D., pp. 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman et al., Nucleic Acids Research 30(1):276-280 (2002)). A set of tools for protein sequence microarray (in silico) analysis is available from the ExPASy proteomics server (Swiss Institute of Bioinformatics (Swiss Institute of Bioinformatics) (Gasteiger et al. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784-3788 (2003). Domains or motifs can also be identified using conventional techniques, eg, by sequence alignment.
关于BRXL多肽，本文中表A3的多肽的比对示于图5。此比对可以用于鉴定在如本文中定义的BRXL多肽之间最保守的结构域或基序。4个这样的结构域是(i)代表BRX结构域的保守结构域1，其包括IPR013591DZC结构域(PFAM登录号PF08381 DZC；由图5中的X标记)；(2)代表BRX结构域的保守结构域2，其包括C端IPR013591DZC结构域(PFAM登录号PF08381 DZC；由图5中的X标记)；(3)保守结构域3和(4)保守结构域4，两者都包含保守Cys，其间隔指示潜在的锌结合基序。全部4个保守结构域在图5中加框指示。With regard to BRXL polypeptides, an alignment of the polypeptides of Table A3 herein is shown in FIG. 5 . This alignment can be used to identify the most conserved domains or motifs among BRXL polypeptides as defined herein. Four such domains are (i) representing the conserved
为比较而进行序列比对的方法是本领域公知的，此类方法包括GAP、BESTFIT、BLAST、FASTA和TFASTA。GAP使用Needleman和Wunsch的算法((1970)J.Mol.Biol.48：443-453)来寻找两序列之间匹配数最大化且空位数最小化的全局(即跨越完整序列)的比对。BLAST算法(Altschul等(1990)J Mol Biol 215：403-10)计算序列同一性百分比，并对两序列之间的相似性进行统计学分析。执行BLAST分析的软件可通过美国国家生物技术信息中心(NCBI)公开地获得。同源物可以例如，使用ClustalW多重序列比对算法(1.83版)，采用默认的成对比对参数以及百分比的记分方法而容易地鉴定。利用可获自MatGAT软件包(Campanella等，(2003)BMCBioinformatics.10：29.2003Jul 10；4：29.MatGAT：an application thatgenerates similarity/identity matrices using protein or DNA sequences)的方法之一，也可以确定全局相似性和同一性百分比。可以进行微小的人工编辑以优化保守基序之间的比对，这对于所属领域的技术人员而言将是显而易见的。此外，除了利用全长序列进行同源物鉴定以外，还可以利用特定的结构域。可以利用上述程序采用默认参数针对完整核酸或氨基酸序列或者选择的结构域或保守基序来确定序列同一性值。对于局部比对，Smith-Waterman算法是特别有用的(Smith TF，Waterman MS(1981)J.Mol.Biol 147(1)；195-7)。Methods of aligning sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48:443-453) to find a global (ie, spanning the entire sequence) alignment between two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215:403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (NCBI). Homologues can be readily identified, for example, using the ClustalW multiple sequence alignment algorithm (version 1.83) with default pairwise alignment parameters and a scoring method of percentages. It is also possible to determine global Similarity and identity percentages. Minor manual editing can be made to optimize the alignment between conserved motifs, as will be apparent to those skilled in the art. Furthermore, in addition to using the full-length sequence for homologue identification, specific domains can also be used. Sequence identity values can be determined for complete nucleic acid or amino acid sequences or selected domains or conserved motifs using the programs described above using default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).
关于BRXL多肽，本文中实施例部分在表B1中描述了SEQ ID NO：18所示的BRXL多肽与表A3所列的BRXL多肽之间的百分比同一性，其可以低至43％氨基酸序列同一性。在一些情况下，可调整缺省参数来改变搜索的严格性。例如使用BLAST，可增加用于报告相对于数据库序列的匹配的统计学显著性阈值(称为“期望”值)，以显示不太严格的匹配。这样，可鉴定到短的几乎完全的匹配。Regarding BRXL polypeptides, the Examples section herein describes in Table B1 the percent identity between the BRXL polypeptide shown in SEQ ID NO: 18 and the BRXL polypeptides listed in Table A3, which can be as low as 43% amino acid sequence identity . In some cases, the default parameters can be adjusted to change the stringency of the search. For example using BLAST, the statistical significance threshold (termed the "expectation" value) for reporting a match against a database sequence can be increased to reveal less stringent matches. In this way, short almost perfect matches can be identified.
预测蛋白质亚细胞定位的任务非常重要并进行了充分研究。知晓蛋白质的定位有助于阐释其功能。用于蛋白质定位的实验方法范围广泛，从免疫定位到利用绿色荧光蛋白(GFP)或β-葡糖醛酸糖苷酶(GUS)标记蛋白质。与计算方法相比，此类方法虽然劳动量大但精确。最近在根据序列数据计算预测蛋白质定位方面取得了很大进步。从瑞士生物信息学研究所托管的ExPASy蛋白质组学工具可获得本领域技术人员公知的算法，例如PSort、TargetP、ChloroP、LocTree、Predotar、LipoP、MITOPROT、PATS、PTS1、SignalP、TMHMM和其他。The task of predicting protein subcellular localization is important and well studied. Knowing where a protein is located helps elucidate its function. Experimental methods for protein localization range from immunolocalization to labeling proteins with green fluorescent protein (GFP) or β-glucuronidase (GUS). Compared with computational methods, such methods are labor-intensive but precise. Much progress has recently been made in computationally predicting protein localization from sequence data. Algorithms known to those skilled in the art such as PSort, TargetP, ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM and others are available from the ExPASy proteomics tool hosted by the Swiss Bioinformatics Institute.
关于BRXL多肽，根据TargetP，SEQ ID NO：18所示的BRXL多肽的预测的亚细胞定位是细胞核(参见实施例部分)。Regarding the BRXL polypeptide, according to TargetP, the predicted subcellular location of the BRXL polypeptide shown in SEQ ID NO: 18 is the nucleus (see the Examples section).
Alfin样多肽，当在植物中，特别地在稻植物中表达时，赋予此类植物增强的对非生物胁迫的耐受性。Alfin-like polypeptides, when expressed in plants, in particular rice plants, confer on such plants enhanced tolerance to abiotic stress.
此外，YRP多肽，当在植物中，特别地在稻植物中表达时，赋予此类植物增强的对非生物胁迫的耐受性。Furthermore, YRP polypeptides, when expressed in plants, particularly in rice plants, confer on such plants enhanced tolerance to abiotic stress.
此外，silky-1样多肽，当在植物中，特别地在稻植物中表达时，赋予此类植物增强的对非生物胁迫的耐受性。Furthermore, silky-1-like polypeptides, when expressed in plants, particularly in rice plants, confer on such plants enhanced tolerance to abiotic stress.
此外，用于测量ARP6多肽的活性的工具和技术在本领域是公知的(Carmona等2007)，如例如本文中包括的文献中描述的。此外，ARP6多肽，当如实施例部分中所述的按照本发明方法在稻中表达时，产生具有增强的产量相关性状的植物，特别是在种子重量、收获指数、种子饱满率和每株植物的饱满种子数之任一或多个方面的增加。Furthermore, tools and techniques for measuring the activity of ARP6 polypeptides are well known in the art (Carmona et al. 2007), as described, for example, in the literature included herein. Furthermore, the ARP6 polypeptide, when expressed in rice according to the methods of the invention as described in the Examples section, gives plants having enhanced yield-related traits, in particular in terms of seed weight, harvest index, seed filling rate and yield per plant An increase in one or more aspects of the number of filled seeds.
再有，POP多肽(至少其天然形式)通常具有内肽酶活性，在脯氨酸残基之后切割，以及较小程度地在Ala残基之后切割。用于测量脯氨酰内肽酶活性的工具和技术在本领域内是公知的，参见例如Nomura(FEBSLetters 209，235-237，1986)。更详细的内容提供于实施例部分。此外，POP多肽，当如实施例部分中所述的按照本发明方法在稻中表达时，产生具有增强的产量相关性状的植物，特别是增加的生物量和/或增加的种子产量。Also, POP polypeptides (at least in their native form) generally have endopeptidase activity, cleave after proline residues, and to a lesser extent after Ala residues. Tools and techniques for measuring prolyl endopeptidase activity are well known in the art, see eg Nomura (
再有，CRL多肽，当存在于植物中时，通常具有调节光合作用的活性。用于测量对光化合作用的调节的工具和技术在本领域是公知的，例如通过测定叶绿素含量或叶绿素荧光来测量(Asano等2004，The Plant Journal，第38卷，pp.448-459(12)。此外，CRL多肽，当如实施例部分中所述的按照本发明方法在稻中表达时，产生具有增强的产量相关性状(特别是在选自种子总产量((Totalwgseeds)、饱满种子数(nrfilledseed)、饱满率(fillrate)和收获指数(harvestindex)之任一或多个方面)的植物。Furthermore, CRL polypeptides, when present in plants, generally have photosynthesis-modulating activity. Tools and techniques for measuring regulation of photosynthesis are well known in the art, for example by measuring chlorophyll content or chlorophyll fluorescence (Asano et al. 2004, The Plant Journal, vol. 38, pp.448-459( 12). In addition, CRL polypeptides, when expressed in rice according to the method of the present invention as described in the Examples section, produce yield-related traits with enhancement (particularly in the group selected from total seed yield ((Totalwgseeds), full seed Number (nrfilledseed), filling rate (fillrate) and harvest index (harvestindex) any one or more aspects) of the plant.
涉及alfin样多肽，可以例如通过用编码SEQ ID NO：2的多肽序列的SEQ ID NO：1或编码SEQ ID NO：4的多肽序列的SEQ ID NO：3的任一个所示的核酸序列转化植物来实施本发明。然而，本发明的实施不限于这些序列；本发明的方法可有利地使用如本文中定义的任何alfin样多肽编码核酸或alfin样多肽来实施。Relating to alfin-like polypeptides, plants can be transformed, for example, by any one of the nucleic acid sequences shown in SEQ ID NO: 1 of the polypeptide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 3 encoding the polypeptide sequence of SEQ ID NO: 4 to implement the present invention. However, the practice of the invention is not limited to these sequences; the methods of the invention can advantageously be carried out using any alfin-like polypeptide-encoding nucleic acid or alfin-like polypeptide as defined herein.
编码alfin样多肽的核酸的实例在本文实施例部分表A1中给出。这样的核酸可用于实施本发明的方法。表A1所给出的氨基酸序列的直向同源物和旁系同源物可使用常规工具和技术例如交互BLAST搜索容易地获得。通常，这包括一次BLAST，即以查询序列(例如，利用实施例部分表A1中所列的任何序列)针对任何序列数据库如可公共获得的NCBI数据库进行BLAST。当从核苷酸序列开始时，通常使用BLASTN或TBLASTX(利用标准默认值)，而当从蛋白质序列开始时，则使用BLASTP或TBLASTN(利用标准默认值)。BLAST结果可以任选地过滤。接着使用过滤的结果或者未过滤的结果中的全长序列针对查询序列来源生物的序列进行反向BLAST(二次BLAST)(在查询序列为SEQ ID NO：1或SEQ ID NO：2的情况下，二次BLAST因此将会针对番茄(Solanum lycopersicum)序列进行，在查询序列为SEQ ID NO：3或SEQ ID NO：4的情况下，二次BLAST因此将会针对毛果杨(Populus trichocarpa)序列进行)。然后比较一次和二次BLAST的结果。如果一次BLAST中分值靠前的命中事件来自查询序列源自的相同物种，而理想地反向BLAST导致查询序列在最高命中事件中，则鉴定到了旁系同源物；如果一次BLAST中分值靠前的命中事件不是来自查询序列源自的相同物种，且优选地反向BLAST导致查询序列处于最高命中事件之列，则找到了直向同源物。Examples of nucleic acids encoding alfin-like polypeptides are given in Table A1 of the Examples section herein. Such nucleic acids are useful in practicing the methods of the invention. Orthologues and paralogues of the amino acid sequences given in Table Al can be readily obtained using conventional tools and techniques such as reciprocal BLAST searches. Typically, this involves a BLAST with the query sequence (eg, using any of the sequences listed in Table A1 of the Examples section) against any sequence database such as the publicly available NCBI database. Typically BLASTN or TBLASTX (using standard defaults) is used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard defaults) when starting from a protein sequence. BLAST results can optionally be filtered. Then use the filtered results or the full-length sequences in the unfiltered results to perform reverse BLAST (secondary BLAST) against the sequence of the source organism of the query sequence (in the case of the query sequence being SEQ ID NO: 1 or SEQ ID NO: 2 , the secondary BLAST will therefore be performed against the tomato (Solanum lycopersicum) sequence, and in the case of the query sequence being SEQ ID NO: 3 or SEQ ID NO: 4, the secondary BLAST will therefore be performed against the Populus trichocarpa sequence conduct). Then compare the results of primary and secondary BLAST. A paralogue is identified if the top-scoring hits in a BLAST are from the same species from which the query sequence originated, and ideally an inverse BLAST results in the query sequence being among the top hits; if a BLAST score An ortholog is found when the top hits are not from the same species from which the query sequence was derived, and preferably BLASTing inverse results in the query sequence being among the top hits.
关于YRP多肽，可以例如通过用编码SEQ ID NO：11的多肽序列的SEQ ID NO：10或编码SEQ ID NO：13的多肽序列的SEQ ID NO：12的任一个所示的核酸序列转化植物来实施本发明。然而，本发明的实施不限于这些序列；本发明的方法可有利地使用如本文中定义的任意YRP多肽编码核酸或YRP多肽来实施。Regarding the YRP polypeptide, plants can be transformed, for example, by any one of the nucleic acid sequence shown in SEQ ID NO: 10 of the polypeptide sequence encoding SEQ ID NO: 11 or SEQ ID NO: 12 encoding the polypeptide sequence of SEQ ID NO: 13. Implement the invention. However, the practice of the invention is not limited to these sequences; the methods of the invention can advantageously be carried out using any YRP polypeptide-encoding nucleic acid or YRP polypeptide as defined herein.
编码YRP多肽的核酸的实例在本文实施例部分表A2中给出。这样的核酸可用于实施本发明的方法。表A2所给出的氨基酸序列的直向同源物和旁系同源物可使用常规工具和技术例如交互BLAST搜索容易地获得。通常，这包括一次BLAST，即以查询序列(例如，利用实施例部分表A2中所列的任何序列)针对任何序列数据库如可公共获得的NCBI数据库进行BLAST。当从核苷酸序列开始时，通常使用BLASTN或TBLASTX(利用标准默认值)，而当从蛋白质序列开始时，则使用BLASTP或TBLASTN(利用标准默认值)。BLAST结果可以任选地过滤。接着使用过滤的结果或者未过滤的结果中的全长序列针对查询序列来源生物的序列进行反向BLAST(二次BLAST)(在查询序列为SEQ ID NO：10或SEQ ID NO：11的情况下，二次BLAST因此将会针对大麦(Hordeum vulgare)序列进行，在查询序列为SEQ ID NO：12或SEQ ID NO：13的情况下，二次BLAST因此将会针对大麦序列进行)。然后比较一次和二次BLAST的结果。如果一次BLAST中分值靠前的命中事件来自查询序列源自的相同物种，而理想地反向BLAST导致查询序列在最高命中事件中，则鉴定到了旁系同源物；如果一次BLAST中分值靠前的命中事件不是来自查询序列源自的相同物种，且优选地反向BLAST导致查询序列处于最高命中事件之列，则找到了直向同源物。Examples of nucleic acids encoding YRP polypeptides are given in Table A2 of the Examples section herein. Such nucleic acids are useful in practicing the methods of the invention. Orthologues and paralogues of the amino acid sequences given in Table A2 can be readily obtained using conventional tools and techniques such as reciprocal BLAST searches. Typically, this involves a BLAST with the query sequence (eg, using any of the sequences listed in Table A2 of the Examples section) against any sequence database such as the publicly available NCBI database. Typically BLASTN or TBLASTX (using standard defaults) is used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard defaults) when starting from a protein sequence. BLAST results can optionally be filtered. Then use the filtered results or the full-length sequences in the unfiltered results to perform reverse BLAST (secondary BLAST) against the sequence of the source organism of the query sequence (in the case of the query sequence being SEQ ID NO: 10 or SEQ ID NO: 11 , the secondary BLAST will thus be performed on the barley (Hordeum vulgare) sequence, in the case of the query sequence being SEQ ID NO: 12 or SEQ ID NO: 13, the secondary BLAST will therefore be performed on the barley sequence). Then compare the results of primary and secondary BLAST. A paralogue is identified if the top-scoring hits in a BLAST are from the same species from which the query sequence originated, and ideally an inverse BLAST results in the query sequence being among the top hits; if a BLAST score An ortholog is found when the top hits are not from the same species from which the query sequence was derived, and preferably BLASTing inverse results in the query sequence being among the top hits.
关于BRXL多肽，可以例如通过用编码SEQ ID NO：18的多肽序列的SEQ ID NO：17所示的核酸序列转化植物来举例说明本发明。然而，本发明的实施不限于这些序列；本发明的方法可有利地使用如本文中定义的任意BRXL多肽编码核酸序列来实施。Regarding the BRXL polypeptide, the present invention can be illustrated, for example, by transforming plants with the nucleic acid sequence shown in SEQ ID NO: 17 encoding the polypeptide sequence of SEQ ID NO: 18. However, the practice of the present invention is not limited to these sequences; the methods of the present invention can advantageously be carried out using any BRXL polypeptide-encoding nucleic acid sequence as defined herein.
编码BRXL多肽的核酸序列的实例在本文实施例1表A3中给出。这样的核酸序列可用于实施本发明的方法。实施例1的表A3所给出的多肽序列为SEQ ID NO：18所示BRXL多肽的直向同源物和旁系同源物的示例序列，术语“直向同源物”和“旁系同源物”如本文所定义。其他的直向同源物和旁系同源物可以通过进行所谓的交互BLAST搜索，容易地找到。通常，这包括一次BLAST，即以查询序列(例如，利用实施例1表A3中所列的任何序列)针对任何序列数据库如可公共获得的NCBI数据库进行BLAST。当从核苷酸序列开始时，通常使用BLASTN或TBLASTX(利用标准默认值)，而当从蛋白质序列开始时，则使用BLASTP或TBLASTN(利用标准默认值)。BLAST结果可以任选地过滤。接着使用过滤的结果或者未过滤的结果中的全长序列针对查询序列来源生物的序列进行反向BLAST(二次BLAST)(在查询序列为SEQ ID NO：17或SEQ ID NO：18的情况下，二次BLAST因此将会针对白杨序列进行)。然后比较一次和二次BLAST的结果。如果一次BLAST中分值靠前的命中事件来自查询序列源自的相同物种，而理想地反向BLAST导致查询序列在最高命中事件中，则鉴定到了旁系同源物；如果一次BLAST中分值靠前的命中事件不是来自查询序列源自的相同物种，且优选地反向BLAST导致查询序列处于最高命中事件之列，则找到了直向同源物。Examples of nucleic acid sequences encoding BRXL polypeptides are given in Table A3 of Example 1 herein. Such nucleic acid sequences can be used to practice the methods of the invention. The polypeptide sequences given in Table A3 of Example 1 are exemplary sequences of orthologs and paralogues of the BRXL polypeptide shown in SEQ ID NO: 18, the terms "orthologs" and "paralogues" "Homologue" is as defined herein. Other orthologues and paralogues can be easily found by performing so-called reciprocal BLAST searches. Typically, this involves a BLAST with the query sequence (eg, using any of the sequences listed in Table A3 of Example 1) against any sequence database such as the publicly available NCBI database. Typically BLASTN or TBLASTX (using standard defaults) is used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard defaults) when starting from a protein sequence. BLAST results can optionally be filtered. Then use the filtered results or the full-length sequences in the unfiltered results to perform reverse BLAST (secondary BLAST) against the sequence of the source organism of the query sequence (in the case of the query sequence being SEQ ID NO: 17 or SEQ ID NO: 18 , a second BLAST will therefore be performed against the poplar sequence). Then compare the results of primary and secondary BLAST. A paralogue is identified if the top-scoring hits in a BLAST are from the same species from which the query sequence originated, and ideally an inverse BLAST results in the query sequence being among the top hits; if a BLAST score An ortholog is found when the top hits are not from the same species from which the query sequence was derived, and preferably BLASTing inverse results in the query sequence being among the top hits.
关于silky-1样多肽，可以例如通过用编码SEQ ID NO：91的多肽序列的SEQ ID NO：90、编码SEQ ID NO：93的多肽序列的SEQ ID NO：92、或编码SEQ ID NO：95的多肽序列的SEQ ID NO：94的任一个所示的核酸序列转化植物来实施本发明。然而，本发明的进行不限定于这些序列；本发明的方法可有利地使用如本文中定义的任意silky-1样多肽编码核酸或silky-1样多肽来实施。Regarding the silky-1-like polypeptide, it can be obtained, for example, by using SEQ ID NO: 90 of the polypeptide sequence encoding SEQ ID NO: 91, SEQ ID NO: 92 of the polypeptide sequence encoding SEQ ID NO: 93, or encoding SEQ ID NO: 95 The nucleic acid sequence shown in any one of SEQ ID NO: 94 of the polypeptide sequence is transformed into a plant to implement the present invention. However, the performance of the invention is not limited to these sequences; the methods of the invention can advantageously be carried out using any silky-1-like polypeptide encoding nucleic acid or silky-1-like polypeptide as defined herein.
编码silky-样多肽的核酸的实例在本文实施例部分表A4中给出。这样的核酸可用于实施本发明的方法。表A4所给出的氨基酸序列的直向同源物和旁系同源物可使用常规工具和技术例如交互BLAST搜索容易地获得。通常，这包括一次BLAST，即以查询序列(例如，利用实施例部分表A4中所列的任何序列)针对任何序列数据库如可公共获得的NCBI数据库进行BLAST。当从核苷酸序列开始时，通常使用BLASTN或TBLASTX(利用标准默认值)，而当从蛋白质序列开始时，则使用BLASTP或TBLASTN(利用标准默认值)。BLAST结果可以任选地过滤。接着使用过滤的结果或者未过滤的结果中的全长序列针对查询序列来源生物的序列进行反向BLAST(二次BLAST)(在查询序列为SEQ ID NO：90或SEQ ID NO：91的情况下，二次BLAST因此将会针对毛果杨序列进行；在查询序列为SEQID NO：92或SEQ ID NO：93的情况下，二次BLAST因此将会针对番茄序列进行；在查询序列为SEQ ID NO：94或SEQ ID NO：95的情况下，二次BLAST因此将会针对小麦(Triticum aestivum)序列进行)。然后比较一次和二次BLAST的结果。如果一次BLAST中分值靠前的命中事件来自查询序列源自的相同物种，而理想地反向BLAST导致查询序列在最高命中事件中，则鉴定到了旁系同源物；如果一次BLAST中分值靠前的命中事件不是来自查询序列源自的相同物种，且优选地反向BLAST导致查询序列处于最高命中事件之列，则找到了直向同源物。Examples of nucleic acids encoding silky-like polypeptides are given in Table A4 of the Examples section herein. Such nucleic acids are useful in practicing the methods of the invention. Orthologues and paralogues of the amino acid sequences given in Table A4 can be readily obtained using conventional tools and techniques such as reciprocal BLAST searches. Typically, this involves a BLAST with the query sequence (eg, using any of the sequences listed in Table A4 of the Examples section) against any sequence database such as the publicly available NCBI database. Typically BLASTN or TBLASTX (using standard defaults) is used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard defaults) when starting from a protein sequence. BLAST results can optionally be filtered. Then use the full-length sequence in the filtered result or the unfiltered result to carry out reverse BLAST (secondary BLAST) against the sequence of the source organism of the query sequence (in the case of the query sequence being SEQ ID NO: 90 or SEQ ID NO: 91 , the secondary BLAST will therefore be performed against the Populus trichocarpa sequence; in the case of the query sequence being SEQ ID NO: 92 or SEQ ID NO: 93, the secondary BLAST will therefore be performed against the tomato sequence; in the case of the query sequence being SEQ ID NO In the case of : 94 or SEQ ID NO: 95, the secondary BLAST will therefore be performed against the wheat (Triticum aestivum) sequence). Then compare the results of primary and secondary BLAST. A paralogue is identified if the top-scoring hits in a BLAST are from the same species from which the query sequence originated, and ideally an inverse BLAST results in the query sequence being among the top hits; if a BLAST score An ortholog is found when the top hits are not from the same species from which the query sequence was derived, and preferably BLASTing inverse results in the query sequence being among the top hits.
关于ARP6多肽，可以例如通过用编码SEQ ID NO：102的多肽序列的SEQ ID NO：101所示的核酸序列转化植物来实施本发明。然而，本发明的实施不限定于这些序列；本发明的方法可有利地使用如本文中定义的任意ARP6多肽编码核酸或ARP6多肽来实施。Regarding the ARP6 polypeptide, the present invention can be implemented, for example, by transforming plants with the nucleic acid sequence shown in SEQ ID NO: 101 encoding the polypeptide sequence of SEQ ID NO: 102. However, the practice of the invention is not limited to these sequences; the methods of the invention can advantageously be carried out using any ARP6 polypeptide-encoding nucleic acid or ARP6 polypeptide as defined herein.
编码ARP6多肽的核酸的实例在本文实施例部分表A5中给出。这样的核酸可用于实施本发明的方法。实施例部分的表A5所给出的氨基酸序列为SEQ ID NO：102所示ARP6多肽的直向同源物和旁系同源物的示例序列，术语“直向同源物”和“旁系同源物”如本文所定义。其他的直向同源物和旁系同源物可以通过进行所谓的交互BLAST搜索，容易地找到。通常，这包括一次BLAST，即以查询序列(例如，利用实施例部分表A5中所列的任何序列)针对任何序列数据库如可公共获得的NCBI数据库进行BLAST。当从核苷酸序列开始时，通常使用BLASTN或TBLASTX(利用标准默认值)，而当从蛋白质序列开始时，则使用BLASTP或TBLASTN(利用标准默认值)。BLAST结果可以任选地过滤。接着使用过滤的结果或者未过滤的结果中的全长序列针对查询序列来源生物的序列进行反向BLAST(二次BLAST)(在查询序列为SEQ ID NO：101或SEQ ID NO：102的情况下，二次BLAST因此将会针对拟南芥(Arabidopsis thaliana)序列进行)。然后比较一次和二次BLAST的结果。如果一次BLAST中分值靠前的命中事件来自查询序列源自的相同物种，而理想地反向BLAST导致查询序列在最高命中事件中，则鉴定到了旁系同源物；如果一次BLAST中分值靠前的命中事件不是来自查询序列源自的相同物种，且优选地反向BLAST导致查询序列处于最高命中事件之列，则找到了直向同源物。Examples of nucleic acids encoding ARP6 polypeptides are given in Table A5 of the Examples section herein. Such nucleic acids are useful in practicing the methods of the invention. The amino acid sequences given in Table A5 of the Examples section are exemplary sequences of orthologues and paralogues of the ARP6 polypeptide shown in SEQ ID NO: 102, the terms "orthologs" and "paralogues" "Homologue" is as defined herein. Other orthologues and paralogues can be easily found by performing so-called reciprocal BLAST searches. Typically, this involves a BLAST with the query sequence (eg, using any of the sequences listed in Table A5 of the Examples section) against any sequence database such as the publicly available NCBI database. Typically BLASTN or TBLASTX (using standard defaults) is used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard defaults) when starting from a protein sequence. BLAST results can optionally be filtered. Then use the filtered results or the full-length sequences in the unfiltered results to perform reverse BLAST (secondary BLAST) against the sequence of the source organism of the query sequence (in the case of the query sequence being SEQ ID NO: 101 or SEQ ID NO: 102 , the secondary BLAST will thus be performed against Arabidopsis thaliana sequences). Then compare the results of primary and secondary BLAST. A paralogue is identified if the top-scoring hits in a BLAST are from the same species from which the query sequence originated, and ideally an inverse BLAST results in the query sequence being among the top hits; if a BLAST score An ortholog is found when the top hits are not from the same species from which the query sequence was derived, and preferably BLASTing inverse results in the query sequence being among the top hits.
关于POP多肽，可以例如通过用编码SEQ ID NO：117的多肽序列的SEQ ID NO：116所示的核酸序列转化植物来举例说明本发明。然而，本发明的进行不限定于这些序列；本发明的方法可有利地使用如本文中定义的任意POP多肽编码核酸或POP多肽来实施。Concerning POP polypeptides, the present invention can be illustrated, for example, by transforming plants with the nucleic acid sequence shown in SEQ ID NO: 116 encoding the polypeptide sequence of SEQ ID NO: 117. However, the performance of the invention is not limited to these sequences; the methods of the invention can advantageously be carried out using any POP polypeptide-encoding nucleic acid or POP polypeptide as defined herein.
编码POP多肽的核酸的实例在本文实施例部分表A6中给出。这样的核酸可用于实施本发明的方法。实施例部分表A6所给出的氨基酸序列为SEQ ID NO：2所示POP多肽的直向同源物和旁系同源物的示例序列，术语“直向同源物”和“旁系同源物”如本文所定义。其他的直向同源物和旁系同源物可以通过进行所谓的交互BLAST搜索，容易地找到。通常，这包括一次BLAST，即以查询序列(例如，利用实施例部分表A6中所列的任何序列)针对任何序列数据库如可公共获得的NCBI数据库进行BLAST。当从核苷酸序列开始时，通常使用BLASTN或TBLASTX(利用标准默认值)，而当从蛋白质序列开始时，则使用BLASTP或TBLASTN(利用标准默认值)。BLAST结果可以任选地过滤。接着使用过滤的结果或者未过滤的结果中的全长序列针对查询序列来源生物的序列进行反向BLAST(二次BLAST)(在查询序列为SEQ ID NO：116或SEQ ID NO：117的情况下，二次BLAST因此将会针对拟南芥序列进行)。然后比较一次和二次BLAST的结果。如果一次BLAST中分值靠前的命中事件来自查询序列源自的相同物种，而理想地反向BLAST导致查询序列在最高命中事件中，则鉴定到了旁系同源物；如果一次BLAST中分值靠前的命中事件不是来自查询序列源自的相同物种，且优选地反向BLAST导致查询序列处于最高命中事件之列，则找到了直向同源物。Examples of nucleic acids encoding POP polypeptides are given in Table A6 of the Examples section herein. Such nucleic acids are useful in practicing the methods of the invention. The amino acid sequences given in Table A6 of the Examples section are exemplary sequences of orthologues and paralogues of the POP polypeptide shown in SEQ ID NO: 2, the terms "orthologs" and "paralogues" "Source" is as defined herein. Other orthologues and paralogues can be easily found by performing so-called reciprocal BLAST searches. Typically, this involves a BLAST with the query sequence (eg, using any of the sequences listed in Table A6 of the Examples section) against any sequence database such as the publicly available NCBI database. Typically BLASTN or TBLASTX (using standard defaults) is used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard defaults) when starting from a protein sequence. BLAST results can optionally be filtered. Then use the full-length sequence in the filtered result or the unfiltered result to carry out reverse BLAST (secondary BLAST) against the sequence of the source organism of the query sequence (in the case of the query sequence being SEQ ID NO: 116 or SEQ ID NO: 117 , a second BLAST will therefore be performed against the Arabidopsis sequence). Then compare the results of primary and secondary BLAST. A paralogue is identified if the top-scoring hits in a BLAST are from the same species from which the query sequence originated, and ideally an inverse BLAST results in the query sequence being among the top hits; if a BLAST score An ortholog is found when the top hits are not from the same species from which the query sequence was derived, and preferably BLASTing inverse results in the query sequence being among the top hits.
关于CRL多肽，可以例如通过用编码SEQ ID NO：156的多肽序列的SEQ ID NO：155所示的核酸序列转化植物来举例说明本发明。然而，本发明的进行不限定于这些序列；本发明的方法可有利地使用如本文中定义的任意CRL多肽编码核酸或CRL多肽来实施。With regard to CRL polypeptides, the present invention can be illustrated, for example, by transforming plants with the nucleic acid sequence shown in SEQ ID NO: 155 encoding the polypeptide sequence of SEQ ID NO: 156. However, the performance of the invention is not limited to these sequences; the methods of the invention can advantageously be carried out using any CRL polypeptide-encoding nucleic acid or CRL polypeptide as defined herein.
编码CRL多肽的核酸的实例在本文实施例部分表A7中给出。这样的核酸可用于实施本发明的方法。实施例部分表A7所给出的氨基酸序列为SEQ ID NO：2所示CRL多肽的直向同源物和旁系同源物的示例序列，术语“直向同源物”和“旁系同源物”如本文所定义。其他的直向同源物和旁系同源物可以通过进行所谓的交互BLAST搜索，容易地找到。通常，这包括一次BLAST，即以查询序列(例如，利用实施例部分表A7中所列的任何序列)针对任何序列数据库如可公共获得的NCBI数据库进行BLAST。当从核苷酸序列开始时，通常使用BLASTN或TBLASTX(利用标准默认值)，而当从蛋白质序列开始时，则使用BLASTP或TBLASTN(利用标准默认值)。BLAST结果可以任选地过滤。接着使用过滤的结果或者未过滤的结果中的全长序列针对查询序列来源生物的序列进行反向BLAST(二次BLAST)(在查询序列为SEQ ID NO：155或SEQ ID NO：156的情况下，二次BLAST因此将会针对拟南芥序列进行)。然后比较一次和二次BLAST的结果。如果一次BLAST中分值靠前的命中事件来自查询序列源自的相同物种，而理想地反向BLAST导致查询序列在最高命中事件中，则鉴定到了旁系同源物；如果一次BLAST中分值靠前的命中事件不是来自查询序列源自的相同物种，且优选地反向BLAST导致查询序列处于最高命中事件之列，则找到了直向同源物。Examples of nucleic acids encoding CRL polypeptides are given in Table A7 of the Examples section herein. Such nucleic acids are useful in practicing the methods of the invention. The amino acid sequences given in Table A7 of the Examples section are exemplary sequences of orthologs and paralogues of the CRL polypeptide shown in SEQ ID NO: 2, the terms "orthologs" and "paralogues" "Source" is as defined herein. Other orthologues and paralogues can be easily found by performing so-called reciprocal BLAST searches. Typically, this involves a BLAST with the query sequence (eg, using any of the sequences listed in Table A7 of the Examples section) against any sequence database such as the publicly available NCBI database. Typically BLASTN or TBLASTX (using standard defaults) is used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard defaults) when starting from a protein sequence. BLAST results can optionally be filtered. Then use the filtered results or the full-length sequence in the unfiltered results to perform reverse BLAST (secondary BLAST) against the sequence of the source organism of the query sequence (in the case of the query sequence being SEQ ID NO: 155 or SEQ ID NO: 156 , a second BLAST will therefore be performed against the Arabidopsis sequence). Then compare the results of primary and secondary BLAST. A paralogue is identified if the top-scoring hits in a BLAST are from the same species from which the query sequence originated, and ideally an inverse BLAST results in the query sequence being among the top hits; if a BLAST score An ortholog is found when the top hits are not from the same species from which the query sequence was derived, and preferably BLASTing inverse results in the query sequence being among the top hits.
分值靠前的命中事件是E值低的命中事件。E值越低，分值越具有显著性(或者换句话说，偶然发现此命中事件的几率越低)。E值的计算是本领域众所周知的。除了E值之外，还可以对比较进行同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在大家族的情况下，可以使用ClustalW，继之以邻接树来辅助对相关基因的聚类进行可视化和鉴定直向同源物和旁系同源物。Hit events with higher scores are hit events with lower E values. The lower the E-value, the more significant the score (or in other words, the lower the chance of finding this hit by chance). Calculation of E-values is well known in the art. In addition to E-values, comparisons can also be scored in percent identity. The percent identity refers to the number of identical nucleotides (or amino acids) over a specific length between two compared nucleic acid (or polypeptide) sequences. In the case of large families, ClustalW followed by a neighbor-joining tree can be used to aid in the visualization of clusters of related genes and the identification of orthologues and paralogues.
核酸变体也可用于实施本发明的方法。这类变体的实例包括编码实施例部分表A1至A7中给出的任一氨基酸序列的同源物和衍生物的核酸，其中“同源物”和“衍生物”如本文所定义。同样可用于本发明方法的有，编码实施例部分表A1至表A7所给出的任一氨基酸序列的直向同源物或旁系同源物的同源物和衍生物的核酸。可用于本发明方法的同源物和衍生物与其源自的未修饰蛋白质具有基本上相同的生物活性和功能活性。用于实施本发明的方法的其他变体可以是其中密码子使用被优化或其中除去了miRNA靶位点的变体。Nucleic acid variants may also be used to practice the methods of the invention. Examples of such variants include nucleic acids encoding homologues and derivatives of any of the amino acid sequences given in Tables A1 to A7 of the Examples section, wherein "homologs" and "derivatives" are as defined herein. Also useful in the methods of the invention are nucleic acids encoding homologues and derivatives of orthologues or paralogues of any one of the amino acid sequences given in Table A1 to Table A7 of the Examples section. Homologues and derivatives useful in the methods of the invention have substantially the same biological and functional activity as the unmodified protein from which they are derived. Other variants for carrying out the methods of the invention may be those in which codon usage is optimized or in which miRNA target sites are removed.
可用于实施本发明方法的其他核酸变体包括编码alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸的部分、与编码alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸杂交的核酸、编码alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸的剪接变体、编码alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸的等位基因变体，以及通过基因改组获得的编码alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸的变体。术语杂交序列、剪接变体、等位基因变体和基因改组如本文所述。Other nucleic acid variants that can be used to practice the methods of the present invention include portions of nucleic acids encoding alfin-like polypeptides or YRP polypeptides or BRXL polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or CRL polypeptides, and encoding alfin-like polypeptides or YRP polypeptides Or BRXL polypeptide or silky-1 sample polypeptide or ARP6 polypeptide or POP polypeptide or CRL polypeptide nucleic acid hybridized nucleic acid, encoding alfin sample polypeptide or YRP polypeptide or BRXL polypeptide or silky-1 sample polypeptide or ARP6 polypeptide or POP polypeptide or CRL polypeptide Splice variants of nucleic acids, allelic variants of nucleic acids encoding alfin-like polypeptides or YRP polypeptides or BRXL polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or CRL polypeptides, as well as encoding alfin-like polypeptides obtained by gene shuffling Or a nucleic acid variant of a YRP polypeptide or a BRXL polypeptide or a silky-1-like polypeptide or an ARP6 polypeptide or a POP polypeptide or a CRL polypeptide. The terms hybridizing sequence, splice variant, allelic variant and gene shuffling are as described herein.
编码alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸无需是全长核酸，因为本发明方法的实施不依赖于全长核酸序列的使用。根据本发明，提供了增强植物的非生物胁迫耐受性的方法，包括在植物中引入和表达实施例部分表A1至A7所给出的任一核酸序列的部分、或者编码实施例部分表A1至A7所给出的任一氨基酸序列的直向同源物、旁系同源物或同源物的核酸的部分。Nucleic acids encoding alfin-like polypeptides or YRP polypeptides or BRXL polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or CRL polypeptides need not be full-length nucleic acids, as performance of the methods of the invention does not rely on the use of full-length nucleic acid sequences. According to the present invention, there is provided a method for enhancing the abiotic stress tolerance of plants, comprising introducing and expressing in plants a part of any nucleic acid sequence given in Table A1 to A7 of the Example Part, or encoding the part of Table A1 of the Example Part Part of the nucleic acid of an orthologue, paralogue or homologue of any of the amino acid sequences given to A7.
例如，可以通过对核酸进行一个或多个缺失来制备核酸的“部分”。“部分”可以以分离的形式使用，或者可将其与其他编码(或非编码)序列融合，以便例如，产生组合了几种活性的蛋白质。当与其他编码序列融合时，经翻译后所产生的多肽可能比针对该蛋白质部分预测的大小要大。For example, a "portion" of a nucleic acid can be prepared by making one or more deletions in the nucleic acid. A "portion" can be used in isolated form, or it can be fused to other coding (or non-coding) sequences, for example, to produce a protein combining several activities. When fused to other coding sequences, the resulting polypeptide may be larger than predicted for the protein portion upon translation.
关于alfin样多肽，可用于本发明方法的部分编码如本文所定义的alfin样多肽，并与实施例部分表A1所给出的氨基酸序列具有基本上相同的生物活性。优选地，“部分”是实施例部分表A1所给出的任一核酸的部分，或是编码实施例部分表A1所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的部分。优选“部分”为长度至少650，700，750，800，850，900，950，1000，1050，1100，1150，1200，1250或更多个连续核苷酸，该连续核苷酸来自实施例部分表A1所给出的任一核酸序列，或编码实施例部分表A1所给出任一氨基酸序列的直向同源物或旁系同源物的核酸。最优选“部分”是SEQ ID NO：1或SEQ ID NO：3的核酸的部分。优选，“部分”编码氨基酸序列的片段，其中所述氨基酸序列，当将其用于系统发生树的构建时，与包含SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列的alfin样多肽组而非与任何其他组聚类。Concerning alfin-like polypeptides, portions useful in the methods of the invention encode alfin-like polypeptides as defined herein and have substantially the same biological activity as the amino acid sequences given in Table A1 of the Examples section. Preferably, a "part" is a portion of any nucleic acid given in Table A1 of the Examples section, or an orthologue or a paralogue of any amino acid sequence given in Table A1 of the Code Examples section part of the nucleic acid. Preferably a "portion" is at least 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250 or more consecutive nucleotides in length from the Examples section Any nucleic acid sequence given in Table A1, or a nucleic acid encoding an orthologue or paralogue of any amino acid sequence given in Table A1 of the Examples section. Most preferably a "portion" is a portion of the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, a "part" encodes a fragment of an amino acid sequence which, when used in the construction of a phylogenetic tree, is similar to an alfin comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 Peptide groups are not clustered with any other groups.
关于YRP多肽，可用于本发明方法的部分编码如本文所定义的YRP多肽，并与实施例部分表A2所给出的氨基酸序列具有基本上相同的生物活性。优选地，“部分”是实施例部分表A2所给出的任一核酸的部分，或是编码实施例部分表A2所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的部分。优选“部分”为长度至少700，750，800，850，900，950，1000，1050，1100，1150，1200，1250，1300，1350，1350，1400或更多个连续核苷酸，该连续核苷酸来自实施例部分表A2所给出的任一核酸序列、或编码实施例部分表A2所给出任一氨基酸序列的直向同源物或旁系同源物的核酸。最优选地，“部分”是SEQ ID NO：10或SEQ ID NO：12的核酸的部分。优选，“部分”编码氨基酸序列的片段，其中所述氨基酸序列，当将其用于系统发生树的构建时，与包含SEQ ID NO：11或SEQ ID NO：13所示的氨基酸序列的YRP多肽的组而非与任何其他组聚类。Concerning YRP polypeptides, portions useful in the methods of the invention encode YRP polypeptides as defined herein and have substantially the same biological activity as the amino acid sequences given in Table A2 of the Examples section. Preferably, a "part" is a portion of any nucleic acid given in Table A2 of the Examples section, or an orthologue or a paralogue encoding any amino acid sequence given in Table A2 of the Examples section part of the nucleic acid. Preferably a "portion" is at least 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1350, 1400 or more contiguous nucleotides in length, the contiguous core The nucleotides are derived from any nucleic acid sequence given in Table A2 of the Examples section, or a nucleic acid encoding an orthologue or paralogue of any amino acid sequence given in Table A2 of the Examples section. Most preferably, a "portion" is a portion of the nucleic acid of SEQ ID NO: 10 or SEQ ID NO: 12. Preferably, "part" encodes a fragment of an amino acid sequence, wherein said amino acid sequence, when used for the construction of a phylogenetic tree, is identical to the YRP polypeptide comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 13 group without clustering with any other group.
关于BRXL多肽，可用于本发明方法的部分编码如本文所定义的BRXL多肽，并与实施例1表A3所给出的多肽序列具有基本上相同的生物活性。优选地，“部分”是实施例1表A3所给出的任一核酸序列的部分，或是编码实施例1表A3所给出的任一多肽序列的直向同源物或旁系同源物的核酸序列的部分。优选“部分”按照递增的优选次序为长度至少600，650，700，750，800，850，900，950，1000，1050，1060，1070，1080或更多个连续核苷酸，该连续核苷酸来自实施例1表A3所给出的任一核酸序列，或编码实施例1表A3所给出任一多肽序列的直向同源物或旁系同源物的核酸序列。优选，所述部分是编码这样的多肽序列的核酸序列的部分，所述多肽序列包含(i)按照递增的优选次序与SEQ ID NO：83所示的保守结构域1(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性；和(ii)按照递增的优选次序与SEQ ID NO：84所示的保守结构域2(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。更优选，所述部分是编码这样的多肽序列的核酸序列的部分，所述多肽序列按照递增的优选次序与SEQ IDNO：18所示的BRXL多肽或与本文中表A3所给出的任一多肽序列具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。最优选，所述部分是SEQ ID NO：17的核酸序列的部分。Concerning BRXL polypeptides, portions useful in the methods of the invention encode BRXL polypeptides as defined herein and have substantially the same biological activity as the polypeptide sequences given in Table A3 of Example 1. Preferably, "part" is a part of any nucleic acid sequence given in Table A3 of Example 1, or an ortholog or paralog of any polypeptide sequence given in Table A3 of Example 1 part of the nucleic acid sequence of the source. Preferably a "portion" is at least 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1060, 1070, 1080 or more consecutive nucleotides in length in increasing order of preference, the consecutive nucleosides The acid is from any nucleic acid sequence given in Table A3 of Example 1, or a nucleic acid sequence encoding an orthologue or a paralogue of any polypeptide sequence given in Table A3 of Example 1. Preferably, said portion is a portion of a nucleic acid sequence encoding a polypeptide sequence comprising (i) at least one of the conserved domain 1 (comprising the BRX domain) shown in SEQ ID NO: 83 in an increasing
关于silky-1样多肽，可用于本发明方法的部分编码如本文所定义的silky-1样多肽，并与实施例部分表A4所给出的氨基酸序列具有基本上相同的生物活性。优选地，“部分”是实施例部分表A4所给出的任一核酸的部分，或是编码实施例部分表A4所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的部分。优选“部分”为长度至少550，600，650，700，750，800，850，900，950，1000，1050，1100或更多个连续核苷酸，该连续核苷酸来自实施例部分表A4所给出的任一核酸序列，或编码实施例部分表A4所给出任一氨基酸序列的直向同源物或旁系同源物的核酸。最优选“部分”是SEQ ID NO：90、SEQ ID NO：92或SEQ ID NO：94的核酸的部分。优选，“部分”编码如下氨基酸序列的片段，其中所述氨基酸序列，当将其用于系统发生树的构建时，与包含SEQ ID NO：91、SEQ ID NO：93或SEQID NO：95所示的氨基酸序列的silky-1样多肽的组而非与任何其他组聚类。Concerning silky-1-like polypeptides, portions useful in the methods of the invention encode silky-1-like polypeptides as defined herein and have substantially the same biological activity as the amino acid sequences given in Table A4 of the Examples section. Preferably, a "part" is a portion of any nucleic acid given in Table A4 of the Examples section, or an orthologue or a paralogue encoding any amino acid sequence given in Table A4 of the Examples section part of the nucleic acid. Preferably a "portion" is at least 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100 or more consecutive nucleotides in length from Table A4 of the Examples section Any of the nucleic acid sequences given, or nucleic acids encoding orthologues or paralogues of any of the amino acid sequences given in Table A4 of the Examples section. Most preferably a "portion" is a portion of the nucleic acid of SEQ ID NO:90, SEQ ID NO:92 or SEQ ID NO:94. Preferably, a "part" encodes a fragment of an amino acid sequence that, when used in the construction of a phylogenetic tree, is identical to that shown in SEQ ID NO: 91, SEQ ID NO: 93 or SEQ ID NO: 95 The group of silky-1-like polypeptides of the amino acid sequence and not clustered with any other group.
关于ARP6多肽，可用于本发明方法的部分编码如本文所定义的ARP6多肽，并与实施例部分表A5所给出的氨基酸序列具有基本上相同的生物活性。优选地，“部分”是实施例部分表A5所给出的任一核酸的部分，或是编码实施例部分表A5所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的部分。优选“部分”为长度至少100、200、300、400、500、550、600、650、700、750、800、850、900、950、1000个连续核苷酸，该连续核苷酸来自实施例部分表A5所给出的任一核酸序列，或编码实施例部分表A5所给出任一氨基酸序列的直向同源物或旁系同源物的核酸。最优选“部分”是SEQ ID NO：101的核酸的部分。优选，“部分”编码氨基酸序列的片段，其中所述氨基酸序列，当将其用于系统发生树例如Kandasamy等2004的图1中描述的系统发生树的构建时，其与包含SEQID NO：102所示的AtARP6的氨基酸序列的ARP6多肽组而非与任何其他组聚类。Concerning ARP6 polypeptides, portions useful in the methods of the invention encode an ARP6 polypeptide as defined herein and have substantially the same biological activity as the amino acid sequences given in Table A5 of the Examples section. Preferably, a "part" is a portion of any nucleic acid given in Table A5 of the Examples section, or an orthologue or a paralogue of any amino acid sequence given in Table A5 of the Code Examples section part of the nucleic acid. Preferably a "portion" is at least 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 consecutive nucleotides in length from the examples Any one of the nucleic acid sequences given in Table A5 of the Examples section, or a nucleic acid encoding an orthologue or paralogue of any of the amino acid sequences given in Table A5 of the Examples section. Most preferably a "portion" is a portion of the nucleic acid of SEQ ID NO: 101. Preferably, a "part" encodes a fragment of an amino acid sequence which, when used in the construction of a phylogenetic tree such as that described in Figure 1 of Kandasamy et al. The group of ARP6 polypeptides for which the amino acid sequence of AtARP6 is shown does not cluster with any other group.
关于POP多肽，可用于本发明方法的部分编码如本文所定义的POP多肽，并与实施例部分表A6所给出的氨基酸序列具有基本上相同的生物活性。优选地，“部分”是实施例部分表A6所给出的任一核酸的部分，或是编码实施例部分表A6所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的部分。优选“部分”为长度至少500、600、700、800、900、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2100、2200、2300个连续核苷酸，该连续核苷酸来自实施例部分表A6所给出的任一核酸序列，或编码实施例部分表A6所给出任一氨基酸序列的直向同源物或旁系同源物的核酸。最优选“部分”是SEQ ID NO：1的核酸的部分。优选，“部分”编码氨基酸序列的片段，其中所述氨基酸序列，当将其用于系统发生树例如图13(Tripathi&Sowdhamini 2006)中描述的系统发生树的构建时，其与箭头所指的包含SEQ ID NO：117所示氨基酸序列的POP多肽组而非与任何其他组聚类。Concerning POP polypeptides, portions useful in the methods of the invention encode a POP polypeptide as defined herein and have substantially the same biological activity as the amino acid sequences given in Table A6 of the Examples section. Preferably, a "part" is part of any nucleic acid given in Table A6 of the Examples section, or an orthologue or paralogue of any amino acid sequence given in Table A6 of the Code Examples section part of the nucleic acid. Preferably a "portion" is at least 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300 consecutive nucleotides in length, The contiguous nucleotides are from any nucleic acid sequence given in Table A6 of the Examples section, or a nucleic acid encoding an orthologue or paralogue of any amino acid sequence given in Table A6 of the Examples section. Most preferably a "portion" is a portion of the nucleic acid of SEQ ID NO:1. Preferably, a "part" encodes a fragment of an amino acid sequence which, when used in the construction of a phylogenetic tree such as that described in Figure 13 (Tripathi & Sowdhamini 2006), contains SEQ The POP polypeptide group of the amino acid sequence shown in ID NO: 117 does not cluster with any other group.
关于CRL多肽，可用于本发明方法的部分编码如本文所定义的CRL多肽，并与实施例部分表A7所给出的氨基酸序列具有基本上相同的生物活性。优选地，“部分”是实施例部分表A7所给出的任一核酸的部分，或是编码实施例部分表A7所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的部分。优选“部分”为长度至少100，200，300，400，500，550，600，650，700，750，800，850，900，950，1000个连续核苷酸，该连续核苷酸来自实施例部分表A7所给出的任一核酸序列、或编码实施例部分表A7所给出任一氨基酸序列的直向同源物或旁系同源物的核酸。最优选“部分”是SEQ ID NO：155的核酸的部分。优选，部分编码氨基酸序列的片段，其中所述氨基酸序列，当将其用于系统发生树例如图16中描述的系统发生树的构建时，其与包含单子叶植物编码的氨基酸序列，更优选双子叶植物编码的序列，最优选SEQ ID NO：156所示的序列，的CRL多肽组而非与任何其他组聚类。Concerning CRL polypeptides, portions useful in the methods of the invention encode a CRL polypeptide as defined herein and have substantially the same biological activity as the amino acid sequences given in Table A7 of the Examples section. Preferably, a "part" is a portion of any nucleic acid given in Table A7 of the Examples section, or an orthologue or a paralogue of any amino acid sequence given in Table A7 of the Code Examples section part of the nucleic acid. Preferably a "portion" is at least 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 consecutive nucleotides in length from the example Partially any of the nucleic acid sequences given in Table A7, or nucleic acids encoding orthologues or paralogues of any of the amino acid sequences given in Partially Table A7. Most preferably a "portion" is a portion of the nucleic acid of SEQ ID NO: 155. Preferably, a fragment that partially encodes an amino acid sequence, wherein said amino acid sequence, when used in the construction of a phylogenetic tree such as the one depicted in Figure 16, is identical to an amino acid sequence comprising a monocotyledonous, more preferably a dicotyledonous The sequence encoded by the leaf plant, most preferably the sequence shown in SEQ ID NO: 156, is not clustered with any other group of CRL polypeptides.
可以用于本发明方法的另一核酸变体是这样的核酸，所述核酸能够在降低的严格条件下，优选在严格条件下，与编码本文中定义的alfin样多肽、或YRP多肽、或BRXL多肽、或silky-1多肽、或ARP6多肽或POP多肽或CRL多肽的核酸，或者与本文中定义的部分杂交。Another nucleic acid variant that can be used in the methods of the present invention is a nucleic acid that is capable of encoding an alfin-like polypeptide as defined herein, or a YRP polypeptide, or a BRXL under reduced stringent conditions, preferably under stringent conditions. A nucleic acid of a polypeptide, or a silky-1 polypeptide, or an ARP6 polypeptide, or a POP polypeptide, or a CRL polypeptide, or hybridizes to a moiety as defined herein.
根据本发明，提供了用于增强植物的非生物胁迫耐受性的方法，包括向植物中引入和表达能够与实施例部分表A1至A7所给出的任一核酸杂交的核酸，或包括向植物中引入和表达能够与编码实施例部分表A1至A7所给出的任何氨基酸序列的直向同源物、旁系同源物或同源物的核酸杂交的核酸。According to the present invention, there is provided a method for enhancing the abiotic stress tolerance of plants, comprising introducing and expressing into plants a nucleic acid capable of hybridizing with any nucleic acid given in Tables A1 to A7 of the Examples section, or comprising introducing Nucleic acids capable of hybridizing to nucleic acids encoding orthologues, paralogues or homologues of any of the amino acid sequences given in Tables A1 to A7 of the Examples section are introduced and expressed in plants.
关于alfin样多肽，可以用于本发明方法的杂交序列编码本文中定义的alfin样多肽，所述多肽与实施例部分表A1所给出的氨基酸序列具有基本上相同的生物活性。优选，杂交序列能够与表A1所给出的任一核酸的互补序列杂交，或与这些序列之任一的部分杂交，其中部分如上文所定义，或者所述杂交序列能够与编码表A1所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的互补序列杂交。最优选，所述杂交序列能够与SEQ ID NO：1或SEQ ID NO：3所示的核酸的互补序列或与其部分杂交。Concerning alfin-like polypeptides, hybridizing sequences that may be used in the methods of the invention encode alfin-like polypeptides as defined herein, which polypeptides have substantially the same biological activity as the amino acid sequences given in Table A1 of the Examples section. Preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of any nucleic acid given in Table A1, or with any part of these sequences, wherein the part is as defined above, or the hybridizing sequence is capable of hybridizing to the coding sequence given in Table A1 Complementary sequence hybridization of nucleic acids of orthologues or paralogues of any amino acid sequence identified. Most preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of the nucleic acid shown in SEQ ID NO: 1 or SEQ ID NO: 3 or to a portion thereof.
优选，杂交序列编码具有氨基酸序列的多肽，其中所述氨基酸序列，当全长用于系统发生树的构建时，其与包含SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列的alfin样多肽的组而非与任何其他组聚类。Preferably, the hybridization sequence encodes a polypeptide having an amino acid sequence, wherein the amino acid sequence, when the full length is used for the construction of a phylogenetic tree, is compatible with an alfin comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 group of polypeptide-like polypeptides rather than clustering with any other group.
关于YRP多肽，可以用于本发明方法的杂交序列编码本文中定义的YRP多肽，所述多肽与实施例部分表A2所给出的氨基酸序列具有基本上相同的生物活性。优选，杂交序列能够与表A2所给出的任一核酸的互补序列杂交，或与这些序列之任一的部分杂交，其中“部分”如上文所定义，或者所述杂交序列能够与编码表A2所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的互补序列杂交。最优选，所述杂交序列能够与SEQ ID NO：10或SEQ ID NO：12所示的核酸的互补序列或与其部分杂交。Concerning YRP polypeptides, hybridizing sequences that may be used in the methods of the invention encode YRP polypeptides as defined herein, which polypeptides have substantially the same biological activity as the amino acid sequences given in Table A2 of the Examples section. Preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of any of the nucleic acids given in Table A2, or to a part of any of these sequences, wherein "part" is as defined above, or said hybridizing sequence is capable of hybridizing to the The nucleic acid complement of an orthologue or paralogue of any given amino acid sequence hybridizes. Most preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of the nucleic acid shown in SEQ ID NO: 10 or SEQ ID NO: 12 or a portion thereof.
优选，杂交序列编码具有氨基酸序列的多肽，其中所述氨基酸序列，当全长用于系统发生树的构建时，其与包含SEQ ID NO：11或SEQ ID NO：13所示的氨基酸序列的YRP多肽的组而非与任何其他组聚类。Preferably, the hybridization sequence encodes a polypeptide having an amino acid sequence, wherein the amino acid sequence, when the full length is used for the construction of a phylogenetic tree, is compatible with YRP comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 13 The group of polypeptides is not clustered with any other group.
关于BRXL多肽，可以用于本发明方法的杂交序列编码本文中定义的BRXL多肽，所述多肽与实施例1表A3所给出的多肽序列具有基本上相同的生物活性。优选，杂交序列能够与实施例1表A3所给出的任一核酸序列或与其互补序列杂交，或与这些序列之任一的部分杂交，其中“部分”如上文所定义，或者其中所述杂交序列能够与编码实施例1表A3所给出的任一多肽序列的直向同源物或旁系同源物的核酸序列或其互补序列杂交。优选，所述杂交序列能够与编码这样的多肽序列的核酸序列杂交，所述多肽序列包含(i)按照递增的优选次序与SEQ ID NO：83所示的保守结构域1(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性；和(ii)按照递增的优选次序与SEQ ID NO：84所示的保守结构域2(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。更优选，所述杂交序列能够与编码这样的多肽序列的核酸序列杂交，所述多肽序列按照递增的优选顺序与SEQ ID NO：18所示的BRXL多肽或本文中表A3所给出的任一多肽序列具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。最优选，杂交序列能够与SEQ ID NO：17所示的核酸序列或其部分杂交。Regarding BRXL polypeptides, hybridizing sequences that can be used in the methods of the present invention encode BRXL polypeptides as defined herein, which have substantially the same biological activity as the polypeptide sequences given in Table A3 of Example 1. Preferably, the hybridizing sequence is capable of hybridizing to any nucleic acid sequence given in Table A3 of Example 1 or its complementary sequence, or to any part of these sequences, wherein "part" is as defined above, or wherein said hybridization The sequence is capable of hybridizing to a nucleic acid sequence encoding an orthologue or paralogue of any of the polypeptide sequences given in Table A3 of Example 1 or its complementary sequence. Preferably, the hybridization sequence is capable of hybridizing to a nucleic acid sequence encoding a polypeptide sequence comprising (i) the conserved domain 1 (comprising the BRX domain) shown in SEQ ID NO: 83 in an increasing preferred order At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity; and (ii) according to An increasing preferred order is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% of the conserved domain 2 (comprising the BRX domain) shown in SEQ ID NO: 84 , 95%, 98%, 99% or higher amino acid sequence identity. More preferably, the hybridizing sequence is capable of hybridizing to a nucleic acid sequence encoding a polypeptide sequence that, in increasing order of preference, is associated with the BRXL polypeptide shown in SEQ ID NO: 18 or any one of the polypeptides shown in Table A3 herein. The polypeptide sequence has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher Amino acid sequence identity. Most preferably, the hybridizing sequence is capable of hybridizing to the nucleic acid sequence shown in SEQ ID NO: 17 or a portion thereof.
关于silky-1样多肽，用于本发明方法的杂交序列编码本文中定义的silky-1样多肽，所述多肽与实施例部分表A4所给出的氨基酸序列具有基本上相同的生物活性。优选，杂交序列能够与表A4所给出的任一核酸的互补序列杂交，或与这些序列之任一的部分杂交，其中“部分”如上文所定义，或者所述杂交序列能够与编码表A4所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的互补序列杂交。最优选，所述杂交序列能够与SEQ ID NO：90、SEQ ID NO：92或SEQ ID NO：94所示的核酸的互补序列或与其部分杂交。Concerning silky-1-like polypeptides, hybridizing sequences used in the methods of the invention encode silky-1-like polypeptides as defined herein, which polypeptides have substantially the same biological activity as the amino acid sequences given in Table A4 of the Examples section. Preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of any nucleic acid given in Table A4, or to a part of any of these sequences, wherein "part" is as defined above, or said hybridizing sequence is capable of hybridizing to a sequence encoding a nucleic acid of Table A4 The nucleic acid complement of an orthologue or paralogue of any given amino acid sequence hybridizes. Most preferably, the hybridizing sequence is capable of hybridizing to the complement of the nucleic acid shown in SEQ ID NO: 90, SEQ ID NO: 92 or SEQ ID NO: 94 or to a portion thereof.
优选，杂交序列编码具有氨基酸序列的多肽，其中所述氨基酸序列，当全长用于系统发生树的构建时，其与包含SEQ ID NO：91、SEQ ID NO：93或SEQ ID NO：95所示的氨基酸序列的silky-1样多肽的组而非与任何其他组聚类。Preferably, the hybridization sequence encodes a polypeptide having an amino acid sequence, wherein the amino acid sequence, when the full length is used for the construction of a phylogenetic tree, is identical to the polypeptide comprising SEQ ID NO: 91, SEQ ID NO: 93 or SEQ ID NO: 95 The group of silky-1-like polypeptides of the amino acid sequence shown is not clustered with any other group.
关于ARP6多肽，用于本发明方法的杂交序列编码本文中定义的ARP6多肽，所述多肽与实施例部分表A5所给出的氨基酸序列具有基本上相同的生物活性。优选，杂交序列能够与实施例部分表A5所给出的任一核酸的互补序列杂交，或与这些序列之任一的部分杂交，其中“部分”如上文所定义，或者所述杂交序列能够与编码实施例部分表A5所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的互补序列杂交。最优选，所述杂交序列能够与SEQ ID NO：101所示的核酸的互补序列或与其部分杂交。Concerning ARP6 polypeptides, hybridizing sequences useful in the methods of the invention encode ARP6 polypeptides as defined herein, said polypeptides having substantially the same biological activity as the amino acid sequences given in Table A5 of the Examples section. Preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of any one of the nucleic acids given in Table A5 of the Examples section, or to a part of any of these sequences, wherein "part" is as defined above, or said hybridizing sequence is capable of hybridizing to Complementary sequences of nucleic acids encoding orthologues or paralogues of any one of the amino acid sequences given in Table A5 of the Examples section hybridize. Most preferably, said hybridizing sequence is capable of hybridizing to the complementary sequence of the nucleic acid shown in SEQ ID NO: 101 or to a portion thereof.
优选，杂交序列编码具有氨基酸序列的多肽，其中所述氨基酸序列，当全长用于系统发生树例如Kandasamy等2004.Trends Plant Sci 9：196-202的图1中描述的系统发生树的构建时，其与包含SEQ ID NO：102所示的AtARP6的氨基酸序列的ARP6多肽组而非与任何其他组聚类。Preferably, the hybridizing sequence encodes a polypeptide having an amino acid sequence, when the full length is used in the construction of a phylogenetic tree such as that described in Figure 1 of Kandasamy et al. 2004. Trends Plant Sci 9: 196-202 , which clusters with the group of ARP6 polypeptides comprising the amino acid sequence of AtARP6 shown in SEQ ID NO: 102 but not with any other group.
关于POP多肽，用于本发明方法的杂交序列编码本文中定义的POP多肽，所述多肽与实施例部分表A6所给出的氨基酸序列具有基本上相同的生物活性。优选，杂交序列能够与实施例部分表A6所给出的任一核酸的互补序列杂交，或与这些序列之任一的部分杂交，其中“部分”如上文所定义，或者所述杂交序列能够与编码实施例部分表A6所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的互补序列杂交。最优选，所述杂交序列能够与SEQ ID NO：116所示的核酸的互补序列或与其部分杂交。Concerning POP polypeptides, hybridizing sequences useful in the methods of the invention encode POP polypeptides as defined herein, which polypeptides have substantially the same biological activity as the amino acid sequences given in Table A6 of the Examples section. Preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of any one of the nucleic acids given in Table A6 of the Examples section, or to a part of any of these sequences, wherein "part" is as defined above, or said hybridizing sequence is capable of hybridizing to Complementary sequences of nucleic acids encoding orthologues or paralogues of any one of the amino acid sequences given in Table A6 of the Examples section hybridize. Most preferably, said hybridizing sequence is capable of hybridizing to the complementary sequence of the nucleic acid shown in SEQ ID NO: 116 or to a portion thereof.
优选，杂交序列编码具有氨基酸序列的多肽，其中所述氨基酸序列，当全长并且用于系统发生树例如图13(Tripathi&Sowdhamini 2006)中描述的系统发生树的构建时，其与箭头指示的包含SEQ ID NO：117所示的氨基酸序列的YRP多肽组而非与任何其他组聚类。Preferably, the hybridizing sequence encodes a polypeptide having an amino acid sequence which, when full-length and used in the construction of a phylogenetic tree such as that described in Figure 13 (Tripathi & Sowdhamini 2006), comprises SEQ The YRP polypeptide group of the amino acid sequence shown in ID NO: 117 does not cluster with any other group.
关于CRL多肽，用于本发明方法的杂交序列编码本文中定义的CRL多肽，所述多肽与实施例部分表A7所给出的氨基酸序列具有基本上相同的生物活性。优选，杂交序列能够与实施例部分表A7所给出的任一核酸的互补序列杂交，或与这些序列之任一的部分杂交，其中“部分”如上文所定义，或者所述杂交序列能够与编码实施例部分表A7所给出的任一氨基酸序列的直向同源物或旁系同源物的核酸的互补序列杂交。最优选，所述杂交序列能够与SEQ ID NO：155所示的核酸的互补序列或与其部分杂交。Concerning CRL polypeptides, hybridizing sequences useful in the methods of the invention encode CRL polypeptides as defined herein, which polypeptides have substantially the same biological activity as the amino acid sequences given in Table A7 of the Examples section. Preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of any one of the nucleic acids given in Table A7 of the Examples section, or to a part of any of these sequences, wherein "part" is as defined above, or said hybridizing sequence is capable of hybridizing to Complementary sequences of nucleic acids encoding orthologues or paralogues of any one of the amino acid sequences given in Table A7 of the Examples section hybridize. Most preferably, the hybridizing sequence is capable of hybridizing to the complementary sequence of the nucleic acid shown in SEQ ID NO: 155 or to a portion thereof.
优选，杂交序列编码具有氨基酸序列的多肽，其中所述氨基酸序列，当全长并且用于系统发生树例如图16中描述的系统发生树的构建时，其与包含单子叶植物编码的氨基酸序列，更优选双子叶植物编码的序列，最优选SEQ ID NO：156所示的序列的CRL多肽的组而非与任何其他组聚类。Preferably, the hybridizing sequence encodes a polypeptide having an amino acid sequence which, when full-length and used in the construction of a phylogenetic tree such as that depicted in Figure 16, is identical to an amino acid sequence comprising a monocot encoding, More preferred are dicot-encoded sequences, most preferably the group of CRL polypeptides of the sequence shown in SEQ ID NO: 156 and not clustered with any other group.
可以用于本发明方法的另一核酸变体是编码前文定义的alfin样多肽、或YRP多肽、或BRXL多肽、或silky-1样多肽、或ARP6多肽、或POP多肽或CRL多肽的剪接变体，剪接变体如本文中定义。Another nucleic acid variant that can be used in the method of the present invention is a splice variant encoding an alfin-like polypeptide, or a YRP polypeptide, or a BRXL polypeptide, or a silky-1-like polypeptide, or an ARP6 polypeptide, or a POP polypeptide or a CRL polypeptide as defined above , a splice variant as defined herein.
根据本发明，提供了用于增强植物的非生物胁迫耐受性和/或增强产量相关性状的方法，包括向植物中引入和表达实施例部分表A1至A7所给出的任一核酸序列的剪接变体、或编码实施例部分表A1至A7所给出的任何氨基酸序列的直向同源物、旁系同源物或同源物的核酸的剪接变体。According to the present invention, there is provided a method for enhancing the abiotic stress tolerance of plants and/or enhancing yield-related traits, comprising introducing and expressing in plants any nucleic acid sequence given in Tables A1 to A7 of the Examples section A splice variant, or a splice variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Tables A1 to A7 of the Examples section.
关于alfin样多肽，优选剪接变体是如SEQ ID NO：1或SEQ ID NO：3所示的核酸的剪接变体、或编码SEQ ID NO：2或SEQ ID NO：4之任一的直向同源物或旁系同源物的核酸的剪接变体。优选，由剪接变体编码的氨基酸序列，当用于系统发生树的构建时，与包含SEQ ID NO：2或SEQ IDNO：4所示氨基酸序列的alfin样多肽的组而非与任何其他组聚类。With regard to alfin-like polypeptides, preferred splice variants are splice variants of nucleic acids as shown in SEQ ID NO: 1 or SEQ ID NO: 3, or encoding any of SEQ ID NO: 2 or SEQ ID NO: 4. A splice variant of a nucleic acid of a homolog or paralog. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, is grouped with an alfin-like polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 rather than with any other kind.
关于YRP多肽，优选剪接变体是如SEQ ID NO：10或SEQ ID NO：12所示的核酸的剪接变体、或编码SEQ ID NO：11或SEQ ID NO：13的直向同源物或旁系同源物的核酸的剪接变体。优选，由剪接变体编码的氨基酸序列，当用于系统发生树的构建时，与包含SEQ ID NO：11或SEQ ID NO：13所示氨基酸序列的YRP多肽的组而非与任何其他组聚类。Regarding the YRP polypeptide, a preferred splice variant is a splice variant of a nucleic acid as shown in SEQ ID NO: 10 or SEQ ID NO: 12, or an ortholog encoding SEQ ID NO: 11 or SEQ ID NO: 13 or A splice variant of a nucleic acid of a paralog. Preferably, the amino acid sequence encoded by the splice variant, when used for the construction of a phylogenetic tree, is grouped with the YRP polypeptide comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 13 rather than with any other group kind.
关于BRXL多肽，优选剪接变体是如SEQ ID NO：17所示的核酸序列的剪接变体、或编码SEQ ID NO：18的直向同源物或旁系同源物的核酸序列的剪接变体。优选，剪接变体是编码这样的多肽序列的核酸序列的剪接变体，所述多肽序列包含(i)按照递增的次序与SEQ ID NO：83所示的保守结构域1(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列；和(ii)按照递增的次序与SEQ ID NO：84所示的保守结构域2(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。更优选，所述剪接变体是编码这样的多肽序列的核酸序列的变体，所述多肽序列按照递增的优选次序与如SEQ IDNO：18所示的BRXL多肽或与本文中表A3所给出的任何多肽序列具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。更优选，剪接变体是如SEQID NO：17所示的核酸序列或编码如SEQ ID NO：18所示的多肽序列的核酸序列的剪接变体。Regarding the BRXL polypeptide, a preferred splice variant is a splice variant of a nucleotide sequence as shown in SEQ ID NO: 17, or a splice variant of a nucleic acid sequence encoding an orthologue or a paralogue of SEQ ID NO: 18 body. Preferably, the splice variant is a splice variant of a nucleic acid sequence encoding a polypeptide sequence comprising (i) the conserved domain 1 (comprising the BRX domain) shown in SEQ ID NO: 83 in increasing order At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more of the amino acid sequence; and (ii) in increasing order The sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the conserved domain 2 (comprising the BRX domain) shown in SEQ ID NO: 84 , 98%, 99% or higher amino acid sequence identity. More preferably, the splice variant is a variant of a nucleic acid sequence encoding a polypeptide sequence that is, in increasing order of preference, related to the BRXL polypeptide shown in SEQ ID NO: 18 or given in Table A3 herein Any polypeptide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher amino acid sequence identity. More preferably, the splice variant is a nucleic acid sequence as shown in SEQ ID NO: 17 or a splice variant of a nucleic acid sequence encoding a polypeptide sequence as shown in SEQ ID NO: 18.
关于silky-1样多肽，优选剪接变体是SEQ ID NO：90、SEQ ID NO：92或SEQ ID NO：94的任何一个所示的核酸的剪接变体，或编码SEQ ID NO：91、SEQ ID NO：93或SEQ ID NO：95的任一个的直向同源物或旁系同源物的核酸的剪接变体。优选，由剪接变体编码的氨基酸序列，当用于系统发生树的构建时，与包含SEQ ID NO：91、SEQ ID NO：93或SEQ ID NO：95所示氨基酸序列的silky-1样多肽的组而非与任何其他组聚类。With regard to silky-1-like polypeptides, preferred splice variants are splice variants of any one of the nucleic acids shown in SEQ ID NO: 90, SEQ ID NO: 92 or SEQ ID NO: 94, or encoding SEQ ID NO: 91, SEQ ID NO: A splice variant of a nucleic acid of an orthologue or paralogue of either of ID NO: 93 or SEQ ID NO: 95. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, is similar to the silky-1-like polypeptide comprising the amino acid sequence shown in SEQ ID NO: 91, SEQ ID NO: 93 or SEQ ID NO: 95 group without clustering with any other group.
关于ARP6多肽，优选剪接变体是如SEQ ID NO：101所示的核酸的剪接变体，或编码SEQ ID NO：102的直向同源物或旁系同源物的核酸的剪接变体。优选，由剪接变体编码的氨基酸序列，当用于系统发生树例如Kandasamy等2004的图1中描述的系统发生树的构建时，与包含SEQ IDNO：102所示的AtARP6的氨基酸序列的ARP6多肽组而非与任何其他组聚类。With regard to the ARP6 polypeptide, a preferred splice variant is a splice variant of a nucleic acid as shown in SEQ ID NO: 101, or a splice variant of a nucleic acid encoding an orthologue or a paralogue of SEQ ID NO: 102. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree such as that described in Figure 1 of Kandasamy et al. 2004, is compatible with an ARP6 polypeptide comprising the amino acid sequence of AtARP6 shown in SEQ ID NO: 102 group rather than clustering with any other group.
关于POP多肽，优选剪接变体是如SEQ ID NO：116所示的核酸的剪接变体，或编码SEQ ID NO：117的直向同源物或旁系同源物的核酸的剪接变体。优选，由剪接变体编码的氨基酸序列，当用于系统发生树例如图13(Tripathi&Sowdhamini 2006)中描述的系统发生树的构建时，与箭头指示的包含SEQ ID NO：117所示氨基酸序列的POP多肽组而非与任何其他组聚类。Regarding the POP polypeptide, a preferred splice variant is a splice variant of a nucleic acid as shown in SEQ ID NO: 116, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 117. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree such as the one described in Figure 13 (Tripathi & Sowdhamini 2006), is associated with the POP indicated by the arrow comprising the amino acid sequence shown in SEQ ID NO: 117 Peptide groups are not clustered with any other groups.
关于CRL多肽，优选剪接变体是如SEQ ID NO：155所示的核酸的剪接变体，或编码SEQ ID NO：156的直向同源物或旁系同源物的核酸的剪接变体。优选，由剪接变体编码的氨基酸序列，当用于系统发生树例如图16中描述的系统发生树的构建时，与包含单子叶植物编码的氨基酸序列，更优选双子叶植物编码的序列，最优选SEQ ID NO：156所示的序列，的CRL多肽组而非与任何其他组聚类。Regarding the CRL polypeptide, a preferred splice variant is a splice variant of a nucleic acid as shown in SEQ ID NO: 155, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 156. Preferably, the amino acid sequences encoded by the splice variants, when used in the construction of a phylogenetic tree such as the one depicted in Figure 16, are identical to those comprising monocot-encoded amino acid sequences, more preferably dicot-encoded sequences, most preferably The group of CRL polypeptides of the sequence shown in SEQ ID NO: 156 is preferred rather than clustered with any other group.
用于实施本发明的方法的另一种核酸变体是编码如上文中描述的alfin样多肽、或YRP多肽、或BRXL多肽、或silky-1样多肽或ARP6多肽、或POP多肽或CRL多肽的核酸的等位基因变体，等位基因变体如本文中定义。Another nucleic acid variant for implementing the method of the present invention is a nucleic acid encoding an alfin-like polypeptide, or a YRP polypeptide, or a BRXL polypeptide, or a silky-1-like polypeptide, or an ARP6 polypeptide, or a POP polypeptide, or a CRL polypeptide as described above The allelic variant of is as defined herein.
根据本发明，提供了增强植物的非生物胁迫耐受性和/或产量相关性状的方法，包括在植物中引入和表达表A1至A7所给出的任一核酸的等位基因变体，或包括在植物中引入和表达编码表A1至A7所给出的任一氨基酸序列的直向同源物、旁系同源物或同源物的核酸的等位基因变体。According to the present invention, there is provided a method for enhancing abiotic stress tolerance and/or yield-related traits of a plant, comprising introducing and expressing in a plant an allelic variant of any nucleic acid given in Tables A1 to A7, or Allelic variants of nucleic acids encoding orthologues, paralogues or homologs of any of the amino acid sequences given in Tables A1 to A7 are included and expressed in plants.
关于alfin样多肽，由可用于本发明方法的等位基因变体编码的多肽与SEQ ID NO：2的alfin样多肽或实施例部分表A1中描述的任一氨基酸序列具有基本上相同的生物活性。等位基因变体天然存在，并且对这些天然等位基因的应用包含于本发明的方法中。优选等位基因变体为SEQ ID NO：1或SEQ ID NO：3的等位基因变体，或编码SEQ ID NO：2或SEQ ID NO：4的直向同源物或旁系同源物的核酸的等位基因变体。优选，由等位基因变体编码的氨基酸序列，与包含SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列的alfin样多肽的组而非与任何其他组聚类。Concerning alfin-like polypeptides, polypeptides encoded by allelic variants useful in the methods of the invention have substantially the same biological activity as the alfin-like polypeptide of SEQ ID NO: 2 or any of the amino acid sequences described in Table A1 of the Examples section . Allelic variants occur naturally, and the use of these natural alleles is encompassed in the methods of the invention. Preferably the allelic variant is an allelic variant of SEQ ID NO: 1 or SEQ ID NO: 3, or encodes an orthologue or paralogue of SEQ ID NO: 2 or SEQ ID NO: 4 Allelic variants of nucleic acids. Preferably, the amino acid sequence encoded by the allelic variant clusters with the group of alfin-like polypeptides comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and not with any other group.
关于YRP多肽，由可用于本发明方法的等位基因变体编码的多肽与SEQ ID NO：11的YRP多肽或实施例部分表A2中描述的任一氨基酸序列具有基本上相同的生物活性。等位基因变体天然存在，并且对这些天然等位基因的应用包含于本发明的方法中。优选等位基因变体为SEQ ID NO：10或SEQ ID NO：12的等位基因变体，或编码SEQ ID NO：11或SEQ IDNO：13的直向同源物或旁系同源物的核酸的等位基因变体。优选，由等位基因变体编码的氨基酸序列，与包含SEQ ID NO：11或SEQ ID NO：13所示的氨基酸序列的YRP多肽的组而非与任何其他组聚类。Concerning YRP polypeptides, polypeptides encoded by allelic variants useful in the methods of the invention have substantially the same biological activity as the YRP polypeptide of SEQ ID NO: 11 or any of the amino acid sequences described in Table A2 of the Examples section. Allelic variants occur naturally, and the use of these natural alleles is encompassed in the methods of the invention. A preferred allelic variant is an allelic variant of SEQ ID NO: 10 or SEQ ID NO: 12, or one encoding an orthologue or paralogue of SEQ ID NO: 11 or SEQ ID NO: 13 An allelic variant of a nucleic acid. Preferably, the amino acid sequence encoded by the allelic variant clusters with the group of YRP polypeptides comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 13 rather than with any other group.
关于BRXL多肽，用于本发明方法的等位基因变体具有与SEQ ID NO：18的BRXL多肽及实施例1表A3中描述的任一多肽序列具有基本上相同的生物活性。等位基因变体天然存在，并且对这些天然等位基因的应用包含于本发明的方法中。优选，等位基因变体是这样的多肽序列的等位基因变体，所述多肽序列包含(i)按照递增的次序与SEQ ID NO：83所示的保守结构域1(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性；和(ii)按照递增的次序与SEQ ID NO：84所示的保守结构域2(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。更优选，所述剪接变体是编码这样的多肽序列的等位基因变体，所述多肽序列按照递增的优选次序与SEQID NO：18所示的BRXL多肽或与本文中表A所给出的任何多肽序列具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。更优选，等位基因变体是SEQ ID NO：17的等位基因变体或编码SEQ ID NO：18的直向同源物或旁系同源物的核酸序列的等位基因变体。With respect to BRXL polypeptides, allelic variants useful in the methods of the invention have substantially the same biological activity as the BRXL polypeptide of SEQ ID NO: 18 and any of the polypeptide sequences described in Table A3 of Example 1. Allelic variants occur naturally, and the use of these natural alleles is encompassed in the methods of the invention. Preferably, the allelic variant is an allelic variant of a polypeptide sequence comprising (i) the conserved domain 1 (comprising the BRX domain) shown in SEQ ID NO: 83 in increasing order At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity; and (ii) according to Incremental order is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher amino acid sequence identity. More preferably, said splice variant is an allelic variant encoding a polypeptide sequence which, in increasing order of preference, is related to the BRXL polypeptide shown in SEQ ID NO: 18 or to the one given in Table A herein Any polypeptide sequence has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher Amino acid sequence identity. More preferably, the allelic variant is an allelic variant of SEQ ID NO: 17 or an allelic variant of a nucleic acid sequence encoding an orthologue or paralogue of SEQ ID NO: 18.
关于silky-1样多肽，由可用于本发明方法的等位基因变体编码的多肽与SEQ ID NO：91的silky-1样多肽或实施例部分表A4中描述的任一氨基酸具有基本上相同的生物活性。等位基因变体天然存在，并且对这些天然等位基因的应用包含于本发明的方法中。优选等位基因变体为SEQ ID NO：90、SEQ ID NO：92或SEQ ID NO：94的任一个的等位基因变体，或编码SEQ ID NO：91、SEQ ID NO：93或SEQ ID NO：95的直向同源物或旁系同源物的核酸的等位基因变体。优选，由等位基因变体编码的氨基酸序列，与包含SEQ ID NO：91、SEQ ID NO：93或SEQ ID NO：95所示的氨基酸序列的silky-1样多肽的组而非与任何其他组聚类。Concerning silky-1-like polypeptides, polypeptides encoded by allelic variants useful in the methods of the invention have substantially the same amino acids as the silky-1-like polypeptide of SEQ ID NO: 91 or any of the amino acids described in Table A4 of the Examples section biological activity. Allelic variants occur naturally, and the use of these natural alleles is encompassed in the methods of the invention. Preferably the allelic variant is an allelic variant of any one of SEQ ID NO: 90, SEQ ID NO: 92 or SEQ ID NO: 94, or encodes SEQ ID NO: 91, SEQ ID NO: 93 or SEQ ID An allelic variant of the nucleic acid of the orthologue or paralogue of NO: 95. Preferably, the amino acid sequence encoded by the allelic variant is grouped with a silky-1-like polypeptide comprising the amino acid sequence shown in SEQ ID NO: 91, SEQ ID NO: 93 or SEQ ID NO: 95 and not with any other group clustering.
关于ARP6多肽，由可用于本发明方法的等位基因变体编码的多肽与SEQ ID NO：102的ARP6多肽及实施例部分表A5中描述的任一氨基酸具有基本上相同的生物活性。等位基因变体天然存在，并且对这些天然等位基因的应用包含于本发明的方法中。优选等位基因变体为SEQ ID NO：101的等位基因变体，或编码SEQ ID NO：102的直向同源物或旁系同源物的核酸的等位基因变体。优选，由等位基因变体编码的氨基酸序列，当用于系统发生树例如Kandasamy等2004.Trends Plant Sci 9：196-202的图1中描述的系统发生树的建立时，与包含SEQ ID NO：102所示的AtARP6的氨基酸序列的ARP6多肽组而非与任何其他组聚类。Concerning ARP6 polypeptides, polypeptides encoded by allelic variants useful in the methods of the invention have substantially the same biological activity as the ARP6 polypeptide of SEQ ID NO: 102 and any of the amino acids described in Table A5 of the Examples section. Allelic variants occur naturally, and the use of these natural alleles is encompassed in the methods of the invention. A preferred allelic variant is an allelic variant of SEQ ID NO: 101, or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 102. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree such as that described in Figure 1 of Kandasamy et al. The ARP6 polypeptide group of the amino acid sequence of AtARP6 shown in :102 does not cluster with any other group.
关于POP多肽，由可用于本发明方法的等位基因变体编码的多肽与SEQ ID NO：117的POP多肽及实施例部分表A6中描述的任一氨基酸具有基本上相同的生物活性。等位基因变体天然存在，并且对这些天然等位基因的应用包含于本发明的方法中。优选等位基因变体为SEQ ID NO：116的等位基因变体，或编码SEQ ID NO：117的直向同源物或旁系同源物的核酸的等位基因变体。优选，由等位基因变体编码的氨基酸序列，当用于系统发生树例如图13(Tripathi&Sowdhamini 2006)中描述的系统发生树的建立时，与由箭头指示的包含SEQ ID NO：117所示的氨基酸序列的POP多肽组而非与任何其他组聚类。Concerning POP polypeptides, polypeptides encoded by allelic variants useful in the methods of the invention have substantially the same biological activity as the POP polypeptide of SEQ ID NO: 117 and any of the amino acids described in Table A6 of the Examples section. Allelic variants occur naturally, and the use of these natural alleles is encompassed in the methods of the invention. A preferred allelic variant is an allelic variant of SEQ ID NO: 116, or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 117. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree such as that described in Figure 13 (Tripathi & Sowdhamini 2006), is identical to the amino acid sequence indicated by the arrow comprising SEQ ID NO: 117. The POP polypeptide group of amino acid sequences is not clustered with any other group.
关于CRL多肽，由可用于本发明方法的等位基因变体编码的多肽与SEQ ID NO：156的CRL多肽及实施例部分表A7中描述的任一氨基酸具有基本上相同的生物活性。等位基因变体天然存在，并且对这些天然等位基因的应用包含于本发明的方法中。优选等位基因变体为SEQ ID NO：155的等位基因变体，或编码SEQ ID NO：156的直向同源物或旁系同源物的核酸的等位基因变体。优选，由等位基因变体编码的氨基酸序列，当用于系统发生树例如图16中描述的系统发生树的建立时，与由单子叶植物编码的氨基酸序列，更优选由双子叶植物编码的序列，最优选由SEQ ID NO：156表示的序列，的CRL多肽组而非与任何其他组聚类。Concerning CRL polypeptides, polypeptides encoded by allelic variants useful in the methods of the invention have substantially the same biological activity as the CRL polypeptide of SEQ ID NO: 156 and any of the amino acids described in Table A7 of the Examples section. Allelic variants occur naturally, and the use of these natural alleles is encompassed in the methods of the invention. A preferred allelic variant is an allelic variant of SEQ ID NO: 155, or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 156. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree such as the one depicted in Figure 16, is identical to the amino acid sequence encoded by a monocot, more preferably a dicot The sequence, most preferably the sequence represented by SEQ ID NO: 156, is not clustered with any other group of CRL polypeptides.
基因改组或定向进化也可用于产生编码上文所定义的alfin样多肽、或者YRP多肽、或者BRXL多肽、或者silky-1样多肽、或者ARP6多肽、或者POP多肽或者CRL多肽的核酸的变体；其中术语“基因改组”如本文所定义。Gene shuffling or directed evolution can also be used to produce the variant of the nucleic acid of the above-defined alfin sample polypeptide, or YRP polypeptide, or BRXL polypeptide, or silky-1 sample polypeptide, or ARP6 polypeptide, or POP polypeptide, or CRL polypeptide; wherein the term "gene shuffling" is as defined herein.
根据本发明，提供了用于增强植物的非生物胁迫耐受性和/或产量相关性状的方法，包括向植物中引入和表达实施例部分表A1至A7所给出的任一核酸序列的变体，或包括向植物中引入和表达编码实施例部分表A1至A7所给出的任何氨基酸序列的直向同源物、旁系同源物或同源物的核酸的变体，其中所述变体核酸序列通过基因改组获得。According to the present invention, there is provided a method for enhancing abiotic stress tolerance and/or yield-related traits of a plant, comprising introducing and expressing a variant of any nucleic acid sequence given in Tables A1 to A7 of the Examples section in a plant variants, or variants comprising the introduction and expression into plants of nucleic acids encoding orthologs, paralogs or homologues of any of the amino acid sequences given in Tables A1 to A7 of the Examples section, wherein said Variant nucleic acid sequences are obtained by gene shuffling.
关于alfin样多肽，优选，由通过基因改组获得的变体核酸编码的氨基酸序列，当用于系统发生树的构建时，与包含SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列的alfin样多肽的组而非与任何其他组聚类。Regarding the alfin-like polypeptide, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used for the construction of a phylogenetic tree, is the same as that comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 The group of alfin-like polypeptides does not cluster with any other group.
关于YRP多肽，优选，由通过基因改组获得的变体核酸编码的氨基酸序列，当用于系统发生树的构建时，与包含SEQ ID NO：11或SEQ ID NO：13所示的氨基酸序列的YRP多肽的组而非与任何其他组聚类。Regarding the YRP polypeptide, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used for the construction of a phylogenetic tree, is the same as the YRP polypeptide comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 13 The group of polypeptides is not clustered with any other group.
关于BRXL多肽，优选，由通过基因改组获得的变体核酸编码这样的多肽序列，所述多肽序列包含(i)按照递增的优选次序与SEQ ID NO：83所示的保守结构域1(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性；和(ii)按照递增的优选次序与SEQ ID NO：84所示的保守结构域2(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。更优选，通过基因改组获得的变体核酸序列编码这样的多肽序列，所述多肽序列按照递增的优选次序与如SEQ ID NO：18所示的BRXL多肽或与本文中表A3所示的任何多肽序列具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。最优选，通过基因改组获得的核酸序列编码如SEQ ID NO：18所示的多肽序列。Regarding the BRXL polypeptide, preferably, the variant nucleic acid obtained by gene shuffling encodes a polypeptide sequence comprising (i) the conserved
关于silky-1样多肽，优选，由通过基因改组获得的变体核酸编码的氨基酸序列，当用于系统发生树的构建时，与包含SEQ ID NO：91、SEQ IDNO：93或SEQ ID NO：95所示的氨基酸序列的silky-1样多肽的组而非与任何其他组聚类。Regarding the silky-1 sample polypeptide, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used for the construction of a phylogenetic tree, contains SEQ ID NO: 91, SEQ ID NO: 93 or SEQ ID NO: The group of silky-1-like polypeptides of the amino acid sequence shown in 95 does not cluster with any other group.
关于ARP6多肽，优选，由通过基因改组获得的变体核酸编码的氨基酸序列，当用于系统发生树例如Kandasamy等2004.Trends Plant Sci 9：196-202的图1中描述的系统发生树的构建时，与包含SEQ ID NO：102所示的AtARP6的氨基酸序列的ARP6多肽组而非与任何其他组聚类。Regarding the ARP6 polypeptide, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used for the construction of a phylogenetic tree such as Kandasamy et al. 2004. Trends Plant Sci 9: 196-202 in Figure 1. , clustered with the ARP6 polypeptide group comprising the amino acid sequence of AtARP6 shown in SEQ ID NO: 102 but not with any other group.
关于POP多肽，优选，由通过基因改组获得的变体核酸编码的氨基酸序列，当用于系统发生树例如图13(Tripathi&Sowdhamini 2006)中描述的系统发生树的构建时，与箭头所指包含SEQ ID NO：117所示的氨基酸序列的POP多肽组而非与任何其他组聚类。Regarding the POP polypeptide, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used for the construction of a phylogenetic tree such as that described in Figure 13 (Tripathi & Sowdhamini 2006), is indicated by the arrow and contains SEQ ID The POP polypeptide group of the amino acid sequence shown in NO: 117 is not clustered with any other group.
关于CRL多肽，优选，由通过基因改组获得的变体核酸编码的氨基酸序列，当用于系统发生树例如图16中描述的系统发生树的构建时，与包含由单子叶植物编码的氨基酸序列，更优选由双子叶植物编码的序列，最优选由SEQ ID NO：156表示的序列，的CRL多肽组而非与任何其他组聚类。Regarding the CRL polypeptide, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used in the construction of a phylogenetic tree such as the phylogenetic tree described in Figure 16, is the same as the amino acid sequence encoded by a monocotyledonous plant, More preferably the group of CRL polypeptides of a sequence encoded by a dicotyledonous plant, most preferably the sequence represented by SEQ ID NO: 156, rather than clustering with any other group.
此外，还可利用定点诱变获得核酸变体。若干方法可用来实现定点诱变，最常见的是基于PCR的方法(Current Protocols in Molecular Biology.Wiley编辑)。In addition, nucleic acid variants can also be obtained using site-directed mutagenesis. Several methods are available to achieve site-directed mutagenesis, the most common being PCR-based methods (Current Protocols in Molecular Biology. Wiley ed.).
编码alfin样多肽的核酸可以源自任何天然或人工来源。该核酸可以通过有意的人为操作而在组成和/或基因组环境上不同于其天然形式。优选，编码alfin样多肽的核酸来自植物，更优选来自单子叶植物或双子叶植物，更优选来自葱属(Allium)或大麦属(Hordeum)植物。。Nucleic acids encoding alfin-like polypeptides may be derived from any natural or artificial source. The nucleic acid may differ from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably, the nucleic acid encoding an alfin-like polypeptide is from a plant, more preferably from a monocotyledonous or dicotyledonous plant, more preferably from a plant of the genus Allium or Hordeum. .
编码YRP多肽的核酸可以源自任何天然或人工来源。该核酸可以通过有意的人为操作修饰而在组成和/或基因组环境上不同于其天然形式。优选，编码YRP多肽的核酸来自植物，更优选来自单子叶植物或双子叶植物，更优选来自杨属(Populus)或茄属(Solanum)植物。Nucleic acids encoding YRP polypeptides may be derived from any natural or artificial source. The nucleic acid may differ from its native form in composition and/or genomic environment through deliberate man-made modifications. Preferably, the nucleic acid encoding the YRP polypeptide is from a plant, more preferably from a monocotyledonous or dicotyledonous plant, more preferably from a plant of the genus Populus or Solanum.
编码BRXL多肽的核酸序列可以源自任何天然或人工来源。该核酸可以通过有意的人为操作修饰而在组成和/或基因组环境上不同于其天然形式。编码BRXL多肽的核酸序列可以来自植物，更优选来自双子叶植物，更优选来自杨柳科(Salicaceae)，最优选核酸序列来自毛果杨。A nucleic acid sequence encoding a BRXL polypeptide may be derived from any natural or artificial source. The nucleic acid may differ from its native form in composition and/or genomic environment through deliberate man-made modifications. The nucleic acid sequence encoding the BRXL polypeptide may be from a plant, more preferably from a dicotyledonous plant, more preferably from the family Salicaceae, most preferably from Populus trichocarpa.
编码silky-1样多肽的核酸可以源自任何天然或人工来源。该核酸可以通过有意的人为操作修饰而在组成和/或基因组环境上不同于其天然形式。优选，编码silky-1样多肽的核酸来自植物，更优选来自单子叶或双子叶植物，更优选来自葱属或大麦属植物。Nucleic acids encoding silky-1-like polypeptides may be derived from any natural or artificial source. The nucleic acid may differ from its native form in composition and/or genomic environment through deliberate man-made modifications. Preferably, the nucleic acid encoding a silky-1-like polypeptide is from a plant, more preferably from a monocotyledonous or dicotyledonous plant, more preferably from a plant of the genus Allium or Hordeum.
编码ARP6多肽的核酸可以源自任何天然或人工来源。该核酸可以通过有意的人为操作修饰而在组成和/或基因组环境上不同于其天然形式。优选，编码ARP6多肽的核酸来自植物，更优选来自双子叶植物，更优选来自十字花科(Brassicaeae)，最优选核酸来自拟南芥。A nucleic acid encoding an ARP6 polypeptide may be derived from any natural or artificial source. The nucleic acid may differ from its native form in composition and/or genomic environment through deliberate man-made modifications. Preferably, the nucleic acid encoding the ARP6 polypeptide is from a plant, more preferably from a dicotyledonous plant, more preferably from the family Brassicaeae, most preferably the nucleic acid is from Arabidopsis thaliana.
编码POP多肽的核酸可以源自任何天然或人工来源。该核酸可以通过有意的人为操作修饰而在组成和/或基因组环境上不同于其天然形式。优选，编码POP多肽的核酸可以来自植物，更优选来自双子叶植物，更优选来自十字花科，最优选核酸来自拟南芥。A nucleic acid encoding a POP polypeptide may be derived from any natural or artificial source. The nucleic acid may differ from its native form in composition and/or genomic environment through deliberate man-made modifications. Preferably, the nucleic acid encoding a POP polypeptide may be from a plant, more preferably from a dicotyledonous plant, more preferably from the family Brassicaceae, most preferably the nucleic acid is from Arabidopsis thaliana.
编码CRL多肽的核酸可以源自任何天然或人工来源。该核酸可以通过有意的人为操作修饰而在组成和/或基因组环境上不同于其天然形式。优选，编码CRL多肽的核酸可以来自植物，更优选来自双子叶植物，更优选来自十字花科，最优选核酸来自拟南芥。Nucleic acids encoding CRL polypeptides can be derived from any natural or artificial source. The nucleic acid may differ from its native form in composition and/or genomic environment through deliberate man-made modifications. Preferably, the nucleic acid encoding a CRL polypeptide may be from a plant, more preferably from a dicotyledonous plant, more preferably from the family Brassicaceae, most preferably the nucleic acid is from Arabidopsis thaliana.
有利地，本发明还提供了迄今未知的CRL编码核酸和CRL多肽。Advantageously, the present invention also provides heretofore unknown CRL-encoding nucleic acids and CRL polypeptides.
根据本发明的再一实施方案，因而提供了分离的核酸分子，其选自：According to yet another embodiment of the present invention, there is thus provided an isolated nucleic acid molecule selected from:
(i)SEQ ID NO：41所示的核酸；(i) the nucleic acid shown in SEQ ID NO: 41;
(ii)SEQ ID NO：41所示的核酸的互补序列；(ii) the complementary sequence of the nucleic acid shown in SEQ ID NO:41;
(iii)编码SEQ ID NO：42所示的多肽的核酸，优选地作为遗传密码简并性的结果，所述分离的核酸可从SEQ ID NO：42所示的多肽序列得到，并且还优选地，赋予相对于对照植物增强的产量相关性状；(iii) a nucleic acid encoding the polypeptide shown in SEQ ID NO: 42, preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be obtained from the polypeptide sequence shown in SEQ ID NO: 42, and also preferably , confers enhanced yield-related traits relative to control plants;
(iv)核酸，其按照递增的优选次序与表A的任何核酸序列具有至少30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、5l％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且还优选地，赋予相对于对照植物增强的产量相关性状；(iv) a nucleic acid having at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 5l%, 52%, 53%, 54%, 55%, 56% , 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and also preferably confers enhanced yield-related traits relative to control plants;
(v)核酸分子，其在严格杂交条件下与(i)至(iv)的核酸分子杂交并且优选地赋予相对于对照植物增强的产量相关性状；(v) nucleic acid molecule, it hybridizes with the nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers the yield-related character that strengthens with respect to control plant;
(vi)编码ASPAT多肽的核酸，所述多肽按照递增的优选次序与SEQID NO：42所示的氨基酸序列以及表A中任何其他氨基酸序列具有至少50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且优选赋予相对于对照植物增强的产量相关性状。(vi) Nucleic acid encoding an ASPAT polypeptide having at least 50%, 51%, 52%, 53%, 54% of the amino acid sequence shown in SEQ ID NO: 42 and any other amino acid sequence in Table A in ascending preferred order %, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and preferably confers enhanced relative to control plants Yield-related traits.
根据本发明的其他实施方案，还提供了分离的多肽，其选自：According to other embodiments of the present invention, there is also provided an isolated polypeptide selected from:
(i)SEQ ID NO：42所示的氨基酸序列；(i) the amino acid sequence shown in SEQ ID NO: 42;
(ii)氨基酸序列，其按照递增的优选次序与SEQ ID NO：42所示的氨基酸序列以及表A中任何其他氨基酸序列具有至少50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且优选赋予相对于对照植物增强的产量相关性状；(ii) an amino acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55% of the amino acid sequence shown in SEQ ID NO: 42 and any other amino acid sequence in Table A in ascending order of preference , 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72 %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and preferably confers enhanced yield-related traits relative to control plants;
(iii)上述(i)或(ii)所给出的任何氨基酸序列的衍生物。(iii) Derivatives of any of the amino acid sequences given in (i) or (ii) above.
关于alfin样多肽、或者YRP多肽、或者silky-1样多肽，本发明的方法的实施产生具有增强的对非生物胁迫的耐受性的植物。Concerning alfin-like polypeptides, or YRP polypeptides, or silky-1-like polypeptides, performance of the methods of the invention gives plants having enhanced tolerance to abiotic stress.
关于BRXL多肽，本发明的方法的实施产生相对于对照植物具有增强的产量相关性状的植物。术语“产量”和“种子产量”更详细地描述于本文中的“定义”部分。Concerning BRXL polypeptides, performance of the methods of the invention gives plants having enhanced yield-related traits relative to control plants. The terms "yield" and "seed yield" are described in more detail in the "Definitions" section herein.
关于ARP6多肽或者CRL多肽，本发明方法的实施产生具有增强的产量相关性状的植物。尤其是，本发明方法的实施产生与对照植物相比较具有增加的产量，特别是增加的种子产量的植物。术语“产量”和“种子产量”在本文“定义”部分有更详细的说明。Concerning ARP6 polypeptides or CRL polypeptides, performance of the methods of the invention gives plants having enhanced yield-related traits. In particular, performance of the methods of the invention gives plants having increased yield, in particular increased seed yield, compared to control plants. The terms "yield" and "seed yield" are described in more detail in the "Definitions" section herein.
关于POP多肽，本发明方法的实施产生具有增强的产量相关性状的植物。尤其是，本发明方法的实施产生与对照植物相比较具有增加的产量，特别是增加的(根和/或枝条)生物量和种子产量的植物。术语“产量”和“种子产量”在本文“定义”部分有更详细的说明。本发明的方法的实施产生具有改变的开花时间(优选更早的开花时间)的植物。Concerning POP polypeptides, performance of the methods of the invention gives plants having enhanced yield-related traits. In particular, performance of the methods of the invention gives plants having increased yield, in particular increased (root and/or shoot) biomass and seed production, compared to control plants. The terms "yield" and "seed yield" are described in more detail in the "Definitions" section herein. Performance of the methods of the invention gives plants with an altered flowering time, preferably an earlier flowering time.
本文中提及增强的产量相关性状旨在表示，植物的一个或多个部分的生物量(重量)的增加，所述部分可以包括地上(可收获的)部分和/或地下(可收获的)部分。特别地，该可收获部分是种子，并且本发明方法的实施导致与对照植物的种子产量相比较具有增加的种子产量的植物。Reference herein to enhanced yield-related traits is intended to mean an increase in the biomass (weight) of one or more parts of a plant, which parts may include above-ground (harvestable) parts and/or below-ground (harvestable) parts part. In particular, the harvestable parts are seeds, and performance of the methods of the invention results in plants having increased seed yield compared to the seed yield of control plants.
以玉米为例，产量增加可以表现为如下一个或多个方面：每平方米建植的植物数的增加、每株植物的穗数的增加、行数、行粒数、粒重、千粒重、穗长度/直径的增加、种子饱满率(为饱满种子数除以种子总数并乘以100)的增加，等等。以稻为例，产量增加可以表现为如下一个或多个方面的增加：每平方米的植物数、每株植物的圆锥花序数、每圆锥花序的小穗数、每圆锥花序的花朵(小花)数(表达为饱满种子数占一级圆锥花序数的比率)、种子饱满率(为饱满种子数除以种子总数并乘以100)的增加、千粒重的增加，等等。Taking corn as an example, an increase in yield can be manifested in one or more of the following aspects: increase in the number of plants per square meter, increase in the number of ears per plant, number of rows, number of grains in a row, grain weight, thousand-grain weight, ear Increase in length/diameter, increase in seed filling rate (number of filled seeds divided by total number of seeds and multiplied by 100), etc. In the case of rice, an increase in yield can be manifested as an increase in one or more of the following: number of plants per square meter, number of panicles per plant, number of spikelets per panicle, flowers (florets) per panicle number (expressed as the ratio of the number of full seeds to the number of first-level panicles), the increase of seed filling rate (the number of full seeds divided by the total number of seeds and multiplied by 100), the increase of thousand-grain weight, and so on.
关于非生物胁迫，本发明提供了相对于对照植物而增强植物的胁迫耐受性的方法，该方法包括调节编码本文所定义的alfin样多肽、或YRP多肽或silky-1多肽的核酸在植物中的表达。With regard to abiotic stress, the present invention provides a method for enhancing the stress tolerance of a plant relative to a control plant, the method comprising modulating in a plant a nucleic acid encoding an alfin-like polypeptide, or a YRP polypeptide or a silky-1 polypeptide as defined herein expression.
通常植物通过更加缓慢的生长来应答胁迫接触。在重度胁迫条件下，植物甚至可以完全停止生长。另一方面，轻度胁迫在文中定义为当植物接触时不导致植物完全停止生长且丧失重新开始生长的能力的任何胁迫。本发明意义上的轻度胁迫导致受胁迫植物的生长，与非胁迫条件下的对照植物相比，下降不到40％、35％、30％或25％、更优选下降不到20％或15％。由于农业实践(灌溉、施肥、农药处理)的发展，栽培的作物植物往往并不会遇到重度胁迫。因此，由轻度胁迫诱发的受损的生长通常成为农业中不期望的性质。轻度胁迫是植物接触的日常的生物和/或非生物(环境)胁迫。非生物胁迫可以因干旱或过量的水、缺氧胁迫、盐胁迫、化学毒性、氧化胁迫以及热、冷或冰冻温度而引起。非生物胁迫可以是由于水胁迫(特别是由于干旱)、盐胁迫、氧化胁迫或离子胁迫引起的渗透胁迫。生物胁迫一般是由病原体例如细菌、病毒、真菌、线虫和昆虫所引起的那些胁迫。Typically plants respond to stress exposure by growing more slowly. Under severe stress conditions, plants can even stop growing altogether. Mild stress, on the other hand, is defined herein as any stress that does not cause the plant to stop growing completely and lose the ability to restart growing when the plant is exposed. Mild stress in the sense of the present invention results in a reduction in the growth of stressed plants by less than 40%, 35%, 30% or 25%, more preferably by less than 20% or 15%, compared to control plants under non-stress conditions. %. Due to the development of agricultural practices (irrigation, fertilization, pesticide treatment), cultivated crop plants often do not experience severe stress. Impaired growth induced by mild stress is therefore generally an undesirable property in agriculture. Mild stress is the daily biotic and/or abiotic (environmental) stress to which plants are exposed. Abiotic stresses can be caused by drought or excess water, anoxic stress, salt stress, chemical toxicity, oxidative stress, and hot, cold or freezing temperatures. Abiotic stress may be osmotic stress due to water stress (especially due to drought), salt stress, oxidative stress or ionic stress. Biological stresses are generally those stresses caused by pathogens such as bacteria, viruses, fungi, nematodes and insects.
特别地，可在(轻度)干旱条件下进行本发明方法以产生相对于对照植物具有增强的干旱耐受性的植物，其本身可表现为相对于对照植物增加的产量。如Wang等(Planta(2003)218：1-14)所报道的那样，非生物胁迫引起一系列的形态学、生理学、生物化学和分子变化，对植物生长和生产力造成不利影响。已知干旱、盐度、极端温度和氧化胁迫相互联系，并可以通过相似的机制诱发生长和细胞损害。Rabbani等(Plant Physiol(2003)133：1755-1767)描述了干旱胁迫和高盐度胁迫之间存在着的特别高程度的“交叉对话”。例如，干旱和/或盐度主要表现为渗透胁迫，导致破坏细胞中的稳态和离子分布。氧化胁迫通常与高温或低温、盐度或干旱胁迫相伴，可以引起功能及结构蛋白质的变性。所以，这些多种多样的环境胁迫通常激活相似的细胞信号传递通路和细胞应答，如应激蛋白的产生、抗氧化剂的上调、可混溶溶质的累积以及生长阻抑。如本文中所用的术语“非胁迫”条件是允许植物最佳生长的那些环境条件。本领域技术人员知道给定位置的正常土壤条件和气候条件。具有最佳生长条件(在非胁迫条件下生长)的植物通常按照递增的优选次序产生这样的植物在给定的环境中的平均产量的至少97％、95％、92％、90％、87％、85％、83％、80％、77％或75％。可基于收获和/或季节，计算平均产量。本领域技术人员将知晓作物的平均产量产出。In particular, the methods of the invention may be performed under (mild) drought conditions to give plants having increased drought tolerance relative to control plants, which may itself manifest as increased yield relative to control plants. As reported by Wang et al. (Planta (2003) 218: 1-14), abiotic stress induces a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, temperature extremes and oxidative stress are known to be linked and can induce growth and cell damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describe a particularly high degree of "cross-talk" between drought stress and high salinity stress. For example, drought and/or salinity primarily manifest as osmotic stress, leading to disruption of homeostasis and ion distribution in cells. Oxidative stress is usually accompanied by high or low temperature, salinity or drought stress, which can cause denaturation of functional and structural proteins. Consequently, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as production of stress proteins, upregulation of antioxidants, accumulation of miscible solutes, and growth arrest. The term "non-stress" conditions as used herein are those environmental conditions which allow optimal growth of plants. Those skilled in the art know the normal soil and climatic conditions for a given location. Plants with optimal growth conditions (grown under non-stress conditions) generally produce at least 97%, 95%, 92%, 90%, 87% of the average yield of such plants in a given environment, in order of increasing preference , 85%, 83%, 80%, 77%, or 75%. Average yields can be calculated based on harvest and/or season. Those skilled in the art will know the average yield yield of a crop.
实施本发明方法可以产生，相对于在相当条件下生长的对照植物，生长在(轻度)干旱条件下具有增强的干旱耐受性的植物。因此，根据本发明，提供了用于在(轻度)干旱条件下生长的植物中增强干旱耐受性的方法，所述方法包括调节编码alfin多肽、或YRP多肽或silky-1样多肽的核酸在植物中的表达。Performance of the methods of the invention may give plants grown under (mild) drought conditions an enhanced drought tolerance relative to control plants grown under comparable conditions. Thus, according to the present invention there is provided a method for increasing drought tolerance in plants grown under (slight) drought conditions, said method comprising modulating a nucleic acid encoding an alfin polypeptide, or a YRP polypeptide or a silky-1-like polypeptide expression in plants.
实施本发明方法可以产生，相对于对照植物，生长在养分缺乏的条件下、特别是氮缺乏条件下对由养分缺乏引起的胁迫具有增强的耐受性的植物。因此，根据本发明，提供了增强对养分缺乏引起的胁迫的耐受性的方法，所述方法包括调节编码alfin样多肽、或YRP多肽、或silky-1样多肽的核酸在植物中的表达。养分缺乏可以因诸如氮、磷酸及其他含磷化合物、钾、钙、镁、锰、铁和硼等养分的缺乏所致。Performance of the method according to the invention can give plants grown under conditions of nutrient deficiency, especially under conditions of nitrogen deficiency, which have an increased tolerance to stress caused by nutrient deficiency relative to control plants. Therefore, according to the present invention, there is provided a method of increasing tolerance to stress caused by nutrient deficiency, said method comprising modulating the expression of a nucleic acid encoding an alfin-like polypeptide, or a YRP polypeptide, or a silky-1-like polypeptide in a plant. Nutrient deficiencies can result from deficiencies in nutrients such as nitrogen, phosphoric acid and other phosphorus-containing compounds, potassium, calcium, magnesium, manganese, iron, and boron.
实施本发明方法可以产生，相对于在相当条件下生长的对照植物，生长在盐胁迫条件下具有增强的盐耐受性的植物。因此，根据本发明，提供了用于在盐胁迫的条件下生长的植物中增强盐耐受性的方法，所述方法包括调节编码alfin样多肽、或YRP多肽、或silky-1样多肽的核酸在植物中的表达。术语盐胁迫不局限于食盐(NaCl)，而可以是如下任何一种或多种：NaCl、KCl、LiCl、MgCl2、CaCl2等。Performance of the methods of the invention can give plants grown under salt stress conditions with enhanced salt tolerance relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing salt tolerance in plants grown under conditions of salt stress, said method comprising modulating a nucleic acid encoding an alfin-like polypeptide, or a YRP polypeptide, or a silky-1-like polypeptide expression in plants. The term salt stress is not limited to common salt (NaCl), but may be any one or more of the following: NaCl, KCl, LiCl,MgCl2 , CaCl2, etc.
关于产量相关性状，本发明提供了用于相对于对照植物增加植物的产量相关性状的方法，所述方法包括在植物中增加编码如本文中定义的BRXL多肽的核酸序列的表达。Concerning yield-related traits, the present invention provides methods for increasing yield-related traits in plants relative to control plants, said method comprising increasing expression in a plant of a nucleic acid sequence encoding a BRXL polypeptide as defined herein.
本发明提供了相对于对照植物增加植物的产量，特别地种子产量，的方法，所述方法包括调节编码本文所定义的ARP6多肽或POP多肽或CRL多肽的核酸在植物中的表达。The invention provides a method for increasing yield, in particular seed yield, of a plant relative to control plants, said method comprising modulating expression in a plant of a nucleic acid encoding an ARP6 polypeptide or a POP polypeptide or a CRL polypeptide as defined herein.
由于本发明的转基因植物具有增强的产量相关性状和/或产量，故，相对于对照植物在其生命周期的相应阶段的生长速率而言，这些植物可呈现增加的生长速率(至少在其部分生命周期中)。Since the transgenic plants of the present invention have enhanced yield-related traits and/or yield, these plants may exhibit an increased growth rate (at least in part of their life cycle) relative to the growth rate of control plants at the corresponding stage of their life cycle. cycle).
增加的生长速率可以特异于植物的一个或多个部分(包括种子)，或者可以基本上遍及整株植物。具有增加生长速率的植物可以具有更短的生命周期。植物的生命周期可以理解为指，从成熟干种子生长至植物已经产生类似于起始材料的成熟干种子的阶段所需的时间。此生命周期可以受到诸如萌发速度、早期活力、生长速率、绿度指数、开花时间和种子成熟速度等因素的影响。生长速率的增加可以发生在植物生命周期的一个或多个阶段，或者发生在基本上整个植物生命周期的过程中。在植物生命周期的早期阶段，生长速率的增加可以反映出增强的活力。生长速率的增加可以改变植物的收获周期，使植物能够比原可能的情况更晚播种和/或更快收获(类似的效果可以通过较早的开花时间获得)。如果生长速率充分增加，可以允许再次播种同种植物物种的种子(例如完全在一个常规的生长期内，播种和收获稻类植物、接着再次播种和收获稻类植物)。与此类似，如果生长速率充分地增加，可以允许再播种不同植物物种的种子(例如播种和收获玉米植物，随后，例如，播种和任选的收获大豆、马铃薯或任何其他适宜的植物)。在一些作物植物的情况下也可能从同一砧木收获增加的次数。改变植物的收获周期可以导致每平方米年生物量产量的增加(这是由于(比方说在一年中)任何特定植物可以生长和收获的次数增加)。与野生型对应物相比，生长速率的增加还允许在更广阔的地域栽培转基因植物，这是因为种植作物的地域限制常由种植时(早季)或收获时(晚季)不利的环境条件所决定。如果缩短收获周期，就可以避免这类不利条件。可以通过自生长曲线获得多种参数，确定生长速率，这类参数可以是：T-Mid(植物达到其最大大小的50％所需的时间)和T-90(植物达到其最大大小的90％所需的时间)等等。The increased growth rate may be specific to one or more parts of the plant (including seeds), or may be throughout substantially the entire plant. Plants with increased growth rates can have shorter life cycles. The life cycle of a plant is understood to mean the time required for growth from mature dry seeds to the stage at which the plant has produced mature dry seeds similar to the starting material. This life cycle can be influenced by factors such as germination rate, early vigor, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth rate can occur at one or more stages of the plant's life cycle, or over the course of substantially the entire plant's life cycle. In the early stages of a plant's life cycle, an increase in growth rate can reflect enhanced vigor. An increase in growth rate can alter a plant's harvest cycle, allowing the plant to be sown later and/or harvested sooner than would otherwise be possible (a similar effect can be achieved with earlier flowering times). If the growth rate is sufficiently increased, re-sowing of seeds of the same plant species may be permitted (eg, sowing and harvesting of rice plants followed by re-sowing and harvesting of rice plants all within one conventional growing period). Similarly, if the growth rate is increased sufficiently, it may allow resowing of seeds of a different plant species (eg, sowing and harvesting of maize plants, followed by, for example, sowing and optional harvesting of soybeans, potatoes, or any other suitable plants). In the case of some crop plants it is also possible to harvest increased times from the same rootstock. Changing the harvest cycle of plants can lead to an increase in annual biomass yield per square meter (due to (say in a year) an increase in the number of times any given plant can be grown and harvested). The increased growth rate also allows the cultivation of transgenic plants over wider territories compared to their wild-type counterparts, since geographical constraints for growing crops are often dictated by unfavorable environmental conditions at planting (early season) or harvesting (late season). decided. Such unfavorable conditions can be avoided if the harvest cycle is shortened. Various parameters can be obtained from the growth curve to determine the growth rate, such parameters can be: T-Mid (the time required for the plant to reach 50% of its maximum size) and T-90 (the time required for the plant to reach 90% of its maximum size time required), etc.
根据本发明优选的方面，实施本发明方法产生相对于对照植物具有增加的生长速率的植物。因此，本发明提供了增加植物生长速率的方法，所述方法包括增加编码本文所定义的BRXL多肽、或ARP6多肽或POP多肽或CRL多肽的核酸在植物中的表达。According to a preferred aspect of the invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Accordingly, the present invention provides a method of increasing the growth rate of plants, said method comprising increasing expression in a plant of a nucleic acid encoding a BRXL polypeptide, or an ARP6 polypeptide, or a POP polypeptide, or a CRL polypeptide as defined herein.
实施本发明方法产生生长在非胁迫条件下或在轻度干旱条件下相对于在相当条件下生长的对照植物具有增加的产量相关性状的植物。因此，根据本发明，提供了用于在非胁迫条件下或在轻度干旱条件下生长的植物中增加产量相关性状的方法，所述方法包括增加编码BRXL多肽或者ARP6多肽或POP多肽或CRL多肽的核酸序列在植物中的表达。Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions having increased yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits in plants grown under non-stress conditions or under mild drought conditions, said method comprising increasing a polypeptide encoding a BRXL polypeptide or an ARP6 polypeptide or a POP polypeptide or a CRL polypeptide. Expression of nucleic acid sequences in plants.
实施本发明方法产生，生长在养分可利用率减小的条件下，尤其是在氮可利用率减小的条件下，相对于在相当条件下生长的对照植物，具有增强的产量相关性状和/或产量的植物。因此，根据本发明，提供了用于在养分可利用率减小的条件下，优选在氮可利用率减小的条件下生长的植物中增加产量相关性状的方法，所述方法包括增加编码BRXL多肽、或ARP6多肽或POP多肽或CRL多肽的核酸序列在植物中的表达。养分可利用率减小可以因养分诸如氮、磷酸及其他含磷化合物、钾、钙、镉、镁、锰、铁和硼等养分的缺乏或过量所致。优选地，养分可利用率减小是氧可利用率减小。Performance of the methods of the invention produces, grown under conditions of reduced nutrient availability, especially under conditions of reduced nitrogen availability, having enhanced yield-related traits and/or compared to control plants grown under comparable conditions or yield of plants. Thus, according to the present invention there is provided a method for increasing yield-related traits in plants grown under conditions of reduced nutrient availability, preferably under conditions of reduced nitrogen availability, said method comprising increasing Expression of the polypeptide, or the nucleic acid sequence of the ARP6 polypeptide, or the POP polypeptide, or the CRL polypeptide in plants. Reduced nutrient availability can result from deficiencies or excesses of nutrients such as nitrogen, phosphoric acid and other phosphorus-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron, and boron. Preferably, the decreased nutrient availability is decreased oxygen availability.
实施本发明方法产生生长在盐胁迫的条件下，相对于在相当条件下生长的对照植物，具有增强的对盐的耐受性的植物。因此，根据本发明，提供了用于在盐胁迫的条件下生长的植物中增强盐耐受性的方法，所述方法包括调节编码ARP6多肽、或POP多肽、或CRL多肽的核酸在植物中的表达。术语盐胁迫不局限于食盐(NaCl)，而可以是如下一种或多种盐：NaCl、KCl、LiCl、MgCl2、CaCl2等。Performance of the methods of the invention gives plants grown under conditions of salt stress having increased tolerance to salt relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for enhancing salt tolerance in plants grown under conditions of salt stress, the method comprising modulating the expression of a nucleic acid encoding an ARP6 polypeptide, or a POP polypeptide, or a CRL polypeptide in a plant. Express. The term salt stress is not limited to common salt (NaCl), but may be one or more of the following: NaCl, KCl, LiCl,MgCl2 ,CaCl2, and the like.
本发明包括可由根据本发明的方法获得的植物或其部分(包括种子)。所述植物或其部分含有有效地连接至在植物中具有功能的启动子的、编码如上文所定义的alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸转基因。The invention includes plants or parts thereof (including seeds) obtainable by a method according to the invention. Said plant or part thereof contains an alfin-like polypeptide as defined above, or a YRP polypeptide or a BRXL polypeptide or a silky-1-like polypeptide or an ARP6 polypeptide or a POP polypeptide or Nucleic acid transgene of CRL polypeptide.
本发明还提供遗传构建体和载体，以利于编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸在植物中的引入和/或表达。可以将基因构建体插入适于转化进入植物并适于在转化的细胞中表达目的基因的载体(可商购获得)中。本发明还提供了如本文所定义的基因构建体在本发明方法中的用途。The present invention also provides genetic constructs and vectors to facilitate the introduction and/or expression of nucleic acids encoding alfin-like polypeptides, or YRP polypeptides, or BRXL polypeptides, or silky-1-like polypeptides, or ARP6 polypeptides, or POP polypeptides, or CRL polypeptides in plants. The gene construct can be inserted into a vector (commercially available) suitable for transformation into plants and for expression of the gene of interest in the transformed cells. The invention also provides the use of a genetic construct as defined herein in the methods of the invention.
更特别地，本发明提供这样的构建体，其含有：More particularly, the present invention provides a construct comprising:
(a)编码如上定义的alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸；(a) coding alfin sample polypeptide as defined above, or the nucleic acid of YRP polypeptide or BRXL polypeptide or silky-1 sample polypeptide or ARP6 polypeptide or POP polypeptide or CRL polypeptide;
(b)一个或多个能够驱动(a)中核酸序列表达的控制序列；和任选的(b) one or more control sequences capable of driving the expression of the nucleic acid sequence in (a); and optionally
(c)转录终止序列。(c) Transcription termination sequence.
优选地，编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸如上文所定义。术语“控制序列”和“终止序列”如本文所定义。Preferably, the nucleic acid encoding an alfin-like polypeptide, or a YRP polypeptide, or a BRXL polypeptide, or a silky-1-like polypeptide, or an ARP6 polypeptide, or a POP polypeptide, or a CRL polypeptide is as defined above. The terms "control sequences" and "termination sequences" are as defined herein.
关于BRXL多肽，优选，构建体的一个控制序列是分离自植物基因组的组成型启动子。植物组成型启动子的一个实例是GOS2启动子，优选来自稻的GOS2启动子，最优选如SEQ ID NO：87所示的GOS2序列。Concerning BRXL polypeptides, preferably, one control sequence of the construct is a constitutive promoter isolated from the plant genome. An example of a plant constitutive promoter is a GOS2 promoter, preferably a GOS2 promoter from rice, most preferably a GOS2 sequence as shown in SEQ ID NO:87.
可以使用含有任何上述核酸的载体转化植物。技术人员充分知晓载体中必须存在的遗传元件，以便成功进行转化、选择并繁殖含目的序列的宿主细胞。目的序列将有效连接于一个或多个控制序列(至少连接于启动子)。Plants can be transformed with vectors containing any of the nucleic acids described above. The skilled artisan is well aware of the genetic elements that must be present in the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest will be operably linked to one or more control sequences (at least to a promoter).
有利地，可以使用任何类型的启动子，无论是天然的还是合成的启动子来驱动核酸序列的表达，但优选启动子是植物来源的。组成型启动子在本发明方法中特别有用。优选组成型启动子也是中等强度的遍在启动子。有关各种启动子类型的定义，参见本文中“定义”部分。Advantageously, any type of promoter, whether natural or synthetic, may be used to drive the expression of the nucleic acid sequence, but preferably the promoter is of plant origin. Constitutive promoters are particularly useful in the methods of the invention. Preferred constitutive promoters are also ubiquitous promoters of moderate strength. See the "Definitions" section herein for definitions of the various promoter types.
关于BRXL多肽，有利地，可以使用任何类型的启动子，无论是天然的还是合成的启动子来增加核酸序列的表达。组成型启动子在本发明方法中特别有用，优选分离自植物基因组的组成型启动子。该植物组成型启动子可以以在所有情况下都比在35S CaMV病毒启动子控制下所获得的水平低的水平驱动编码序列表达。这样的启动子的一个实例是如SEQ ID NO：87所示的GOS2启动子。With regard to BRXL polypeptides, advantageously, any type of promoter, whether natural or synthetic, can be used to increase the expression of the nucleic acid sequence. Constitutive promoters are particularly useful in the methods of the invention, preferably constitutive promoters isolated from plant genomes. This plant constitutive promoter can drive the expression of coding sequences at lower levels in all cases than that obtained under the control of the 35S CaMV viral promoter. An example of such a promoter is the GOS2 promoter shown in SEQ ID NO:87.
在BRXL基因的情况下，器官特异性启动子，例如用于在叶、茎、块茎、分生组织、种子中优先表达的启动子，可用于实施本发明方法。发育调控型和诱导型启动子也可用于实施本发明方法。有关各种启动子类型的定义，参见本文“定义”部分。In the case of the BRXL gene, organ-specific promoters, such as promoters for preferential expression in leaves, stems, tubers, meristems, seeds, can be used to carry out the methods of the invention. Developmentally regulated and inducible promoters can also be used to practice the methods of the invention. See the "Definitions" section herein for definitions of the various promoter types.
关于alfin样多肽，应当清楚，本发明的实施并不局限于SEQ ID NO：1或SEQ ID NO：3所示的alfin样多肽编码核酸，而且本发明的实施也不局限于由组成型启动子所驱动的alfin样多肽编码核酸的表达。With regard to alfin-like polypeptides, it should be clear that the practice of the invention is not limited to nucleic acids encoding alfin-like polypeptides set forth in SEQ ID NO: 1 or SEQ ID NO: 3, nor is the practice of the invention limited to the expression of genes produced by constitutive promoters. The expression of the nucleic acid encoding the alfin-like polypeptide is driven.
所述组成型启动子优选是中等强度的启动子，更优选选自植物来源的启动子例如GOS2启动子，更优选启动子是来自稻的GOS2启动子。甚至更优选，组成型启动子由与SEQ ID NO：7基本上相似的核酸序列表示，最优选组成型启动子如SEQ ID NO：7所示。有关组成型启动子的其他实例参见本文中“定义”部分。The constitutive promoter is preferably a medium-strength promoter, more preferably selected from plant-derived promoters such as the GOS2 promoter, more preferably the promoter is a GOS2 promoter from rice. Even more preferably, the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO:7, most preferably the constitutive promoter is as shown in SEQ ID NO:7. See the "Definitions" section herein for additional examples of constitutive promoters.
任选的，可以在引入植物的构建体中使用一个或多个终止子序列。优选，构建体包含含有与SEQ ID NO：7基本相似的GOS2启动子和编码alfin样多肽的核酸的表达盒。Optionally, one or more terminator sequences may be used in the construct introduced into the plant. Preferably, the construct comprises an expression cassette comprising a GOS2 promoter substantially similar to SEQ ID NO: 7 and a nucleic acid encoding an alfin-like polypeptide.
关于YRP多肽，应当清楚，本发明的实施并不局限于SEQ ID NO：10或SEQ ID NO：12所示的YRP多肽编码核酸序列，而且本发明的实施也不局限于由组成型启动子所驱动的编码YRP多肽的核酸序列的表达。Regarding the YRP polypeptide, it should be clear that the implementation of the present invention is not limited to the YRP polypeptide encoding nucleic acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12, and the implementation of the present invention is not limited to the YRP polypeptide encoded by the constitutive promoter. Driven expression of a nucleic acid sequence encoding a YRP polypeptide.
所述组成型启动子优选是中等强度启动子，更优选选自植物来源的启动子，例如GOS2启动子，更优选是来自稻的GOS2启动子。更优选组成型启动子由与SEQ ID NO：16基本上相似的核酸序列表示，最优选组成型启动子如SEQ ID NO：16所示。有关组成型启动子的其他实例，参见本文中“定义”部分。The constitutive promoter is preferably a medium-strength promoter, more preferably selected from plant-derived promoters, such as a GOS2 promoter, more preferably a GOS2 promoter from rice. A more preferred constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 16, and the most preferred constitutive promoter is shown in SEQ ID NO: 16. For additional examples of constitutive promoters, see the "Definitions" section herein.
任选的，可以在引入植物的构建体中使用一个或多个终止子序列。优选，构建体包含表达盒，所述表达盒包含与SEQ ID NO：16基本上相似的(GOS2)启动子和编码YRP多肽的核酸。Optionally, one or more terminator sequences may be used in the construct introduced into the plant. Preferably, the construct comprises an expression cassette comprising a (GOS2) promoter substantially similar to SEQ ID NO: 16 and a nucleic acid encoding a YRP polypeptide.
关于BRXL多肽，应当清楚，本发明的实施并不局限于SEQ ID NO：17所示的BRXL多肽编码核酸序列，而且本发明的实施也不局限于由组成型启动子所驱动的编码BRXL多肽的核酸序列的表达。With regard to the BRXL polypeptide, it should be clear that the implementation of the present invention is not limited to the BRXL polypeptide-encoding nucleic acid sequence shown in SEQ ID NO: 17, and the implementation of the present invention is not limited to those encoding the BRXL polypeptide driven by a constitutive promoter. Expression of Nucleic Acid Sequences.
任选的，可以在引入植物的构建体中使用一个或多个终止子序列。Optionally, one or more terminator sequences may be used in the construct introduced into the plant.
关于silky-1样多肽，应当清楚，本发明的实施并不局限于SEQ ID NO：90、SEQ ID NO：92或SEQ ID NO：94所示的编码silky-1样多肽的核酸序列，而且本发明的实施也不局限于由组成型启动子所驱动的编码silky-1样多肽的核酸序列的表达。.Regarding the silky-1 sample polypeptide, it should be clear that the implementation of the present invention is not limited to the nucleic acid sequence encoding the silky-1 sample polypeptide shown in SEQ ID NO: 90, SEQ ID NO: 92 or SEQ ID NO: 94, and the present invention Nor is the practice of the invention limited to expression of nucleic acid sequences encoding silky-1-like polypeptides driven by constitutive promoters. .
所述组成型启动子优选是中等强度启动子，更优选选自植物来源的启动子，例如GOS2启动子，更优选是来自稻的GOS2启动子。更优选组成型启动子由与SEQ ID NO：96基本上相似的核酸序列表示，最优选组成型启动子如SEQ ID NO：96所示。有关组成型启动子的其他实例，参见本文中“定义”部分。The constitutive promoter is preferably a medium-strength promoter, more preferably selected from plant-derived promoters, such as a GOS2 promoter, more preferably a GOS2 promoter from rice. A more preferred constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 96, and a most preferred constitutive promoter is shown in SEQ ID NO: 96. For additional examples of constitutive promoters, see the "Definitions" section herein.
任选的，可以在引入植物的构建体中使用一个或多个终止子序列。优选，构建体包含表达盒，所述表达盒包含与SEQ ID NO：96基本上相似的(GOS2)启动子和编码silky-1样多肽的核酸。Optionally, one or more terminator sequences may be used in the construct introduced into the plant. Preferably, the construct comprises an expression cassette comprising a (GOS2) promoter substantially similar to SEQ ID NO: 96 and a nucleic acid encoding a silky-1-like polypeptide.
关于ARP6多肽，应当清楚，本发明的实施并不局限于SEQ ID NO：101所示的ARP6多肽编码核酸，而且本发明的实施也不局限于由组成型启动子所驱动的编码ARP6多肽的核酸的表达。Regarding the ARP6 polypeptide, it should be clear that the practice of the present invention is not limited to the nucleic acid encoding the ARP6 polypeptide shown in SEQ ID NO: 101, and the practice of the present invention is not limited to the nucleic acid encoding the ARP6 polypeptide driven by a constitutive promoter. expression.
所述组成型启动子优选是中等强度启动子，更优选选自植物来源的启动子，例如GOS2启动子，更优选是来自稻的GOS2启动子。更优选组成型启动子由与SEQ ID NO：115基本上相似的核酸序列表示，最优选组成型启动子如SEQ ID NO：115所示。有关组成型启动子的其他实例，参见本文中“定义”部分。The constitutive promoter is preferably a medium-strength promoter, more preferably selected from plant-derived promoters, such as a GOS2 promoter, more preferably a GOS2 promoter from rice. A more preferred constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 115, and the most preferred constitutive promoter is shown in SEQ ID NO: 115. For additional examples of constitutive promoters, see the "Definitions" section herein.
任选的，可以在引入植物的构建体中使用一个或多个终止子序列。优选，构建体包含表达盒，所述表达盒包含与SEQ ID NO：115基本上相似的GOS2启动子和编码ARP6多肽的核酸。Optionally, one or more terminator sequences may be used in the construct introduced into the plant. Preferably, the construct comprises an expression cassette comprising a GOS2 promoter substantially similar to SEQ ID NO: 115 and a nucleic acid encoding an ARP6 polypeptide.
关于POP多肽，应当清楚，本发明的实施并不局限于SEQ ID NO：116所示的POP多肽编码核酸，而且本发明的实施也不局限于由组成型启动子所驱动的编码POP多肽的核酸的表达。With respect to POP polypeptides, it should be clear that the practice of the present invention is not limited to nucleic acids encoding POP polypeptides as set forth in SEQ ID NO: 116, nor is the practice of the present invention limited to nucleic acids encoding POP polypeptides driven by constitutive promoters expression.
所述组成型启动子优选是中等强度启动子，更优选选自植物来源的启动子，例如GOS2启动子，更优选是来自稻的GOS2启动子。更优选组成型启动子由与SEQ ID NO：134基本上相似的核酸序列表示，最优选组成型启动子如SEQ ID NO：134所示。有关组成型启动子的其他实例，参见本文中“定义”部分。The constitutive promoter is preferably a medium-strength promoter, more preferably selected from plant-derived promoters, such as a GOS2 promoter, more preferably a GOS2 promoter from rice. A more preferred constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 134, and a most preferred constitutive promoter is shown in SEQ ID NO: 134. For additional examples of constitutive promoters, see the "Definitions" section herein.
任选的，可以在引入植物的构建体中使用一个或多个终止子序列。优选，构建体包含表达盒，所述表达盒包含与SEQ ID NO：134基本上相似的GOS2启动子和编码POP多肽的核酸。Optionally, one or more terminator sequences may be used in the construct introduced into the plant. Preferably, the construct comprises an expression cassette comprising a GOS2 promoter substantially similar to SEQ ID NO: 134 and a nucleic acid encoding a POP polypeptide.
关于CRL多肽，应当清楚，本发明的实施并不局限于SEQ ID NO：155所示的CRL多肽编码核酸，而且本发明的实施也不局限于由组成型启动子或由根特异性启动子所驱动的CRL多肽编码核酸的表达。Regarding CRL polypeptides, it should be clear that the practice of the present invention is not limited to the CRL polypeptide-encoding nucleic acid shown in SEQ ID NO: 155, nor is the practice of the present invention limited to those produced by a constitutive promoter or by a root-specific promoter. Expression of a nucleic acid encoding a CRL polypeptide is driven.
所述组成型启动子优选是中等强度启动子，更优选选自植物来源的启动子，例如GOS2启动子，更优选是来自稻的GOS2启动子。更优选组成型启动子由与SEQ ID NO：213基本上相似的核酸序列表示，最优选组成型启动子如SEQ ID NO：213所示。有关组成型启动子的其他实例，参见本文中“定义”部分。The constitutive promoter is preferably a medium-strength promoter, more preferably selected from plant-derived promoters, such as a GOS2 promoter, more preferably a GOS2 promoter from rice. A more preferred constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 213, and a most preferred constitutive promoter is shown in SEQ ID NO: 213. For additional examples of constitutive promoters, see the "Definitions" section herein.
任选的，可以在引入植物的构建体中使用一个或多个终止子序列。优选，构建体包含表达盒，所述表达盒包含与SEQ ID NO：213基本上相似的(GOS2)启动子和编码CRL多肽的核酸。Optionally, one or more terminator sequences may be used in the construct introduced into the plant. Preferably, the construct comprises an expression cassette comprising a (GOS2) promoter substantially similar to SEQ ID NO: 213 and a nucleic acid encoding a CRL polypeptide.
另外的调控元件可以包括转录和翻译的增强子。本领域技术人员会知道适合用于实施本发明的终止子和增强子的序列。如“定义”部分所说明的那样，也可以向5’非翻译区(UTR)或在编码序列中加入内含子序列，来增加在胞质中累积的成熟信使的量。其他控制序列(除启动子、增强子、沉默子、内含子序列、3’UTR和/或5’UTR区域之外)可以有蛋白质和/或RNA稳定元件。这类序列为本领域技术人员公知或者可以容易地获得。Additional regulatory elements may include enhancers of transcription and translation. Those skilled in the art will know suitable terminator and enhancer sequences for use in practicing the invention. As explained in the "Definitions" section, intronic sequences can also be added to the 5' untranslated region (UTR) or within the coding sequence to increase the amount of mature message that accumulates in the cytoplasm. Other control sequences (besides promoters, enhancers, silencers, intron sequences, 3'UTR and/or 5'UTR regions) may have protein and/or RNA stabilizing elements. Such sequences are known or readily available to those skilled in the art.
本发明的遗传构建体可以还包括为在特定细胞类型中维持和/或复制所需的复制起点序列。一个实例是需要将遗传构建体作为染色体外遗传元件(如质粒或粘粒分子)在细菌细胞中维持的情况。优选的复制起点包括但不限于f1-ori和colE1。The genetic constructs of the invention may also include origin of replication sequences required for maintenance and/or replication in a particular cell type. An example is where a genetic construct needs to be maintained in a bacterial cell as an extrachromosomal genetic element such as a plasmid or cosmid molecule. Preferred origins of replication include, but are not limited to, f1-ori and colE1.
为检测本发明方法中所用核酸序列的成功转移和/或选择含有这些核酸的转基因植物，最好使用标记基因(或报告基因)。因此，遗传构建体可以任选地含有可选择标记基因。可选择标记在本文“定义”部分有更详细的说明。一旦不再需要标记基因可从转基因细胞将其除去或切除。用于标记去除的技术在本领域内是已知的，有用的技术描述于上文中定义部分。To detect the successful transfer of nucleic acid sequences used in the methods of the invention and/or to select transgenic plants containing these nucleic acids, marker genes (or reporter genes) are preferably used. Thus, a genetic construct may optionally contain a selectable marker gene. Optional markers are described in more detail in the "Definitions" section herein. The marker gene can be removed or excised from the transgenic cell once it is no longer needed. Techniques for label removal are known in the art, useful techniques are described in the definitions section above.
已知对于核酸序列在植物细胞中的稳定或瞬时整合，取决于所用的表达载体和所用的转染技术，仅少数细胞可以摄入外来DNA，以及如果期望的话整合进其基因组。为鉴定并选择这些整合体，通常将编码可选择标记(例如上文所述的那些)的基因与目的基因一起引入宿主细胞中。这些标记能够在例如如下突变体中使用，所述突变体中原有的这些基因例如通过常规方法缺失而丧失功能。此外，编码可选择标记的核酸序列分子可与编码本发明多肽的或用于本发明方法的序列包含在同一个载体中，或者在分开的载体中引入宿主细胞。已经稳定转染了所引入的核酸序列的细胞可以例如通过选择(例如，整合有可选择标记的细胞存活而其他细胞死去)予以鉴定。标记基因一旦不再需要，可以从转基因细胞中除去或切除。用于标记基因去除的技术在本领域公知，有用的技术在上文“定义”部分有说明。It is known that for stable or transient integration of nucleic acid sequences in plant cells, depending on the expression vector used and the transfection technique used, only a few cells can take up foreign DNA and, if desired, integrate into its genome. To identify and select for these integrants, typically a gene encoding a selectable marker (such as those described above) is introduced into the host cell along with the gene of interest. These markers can be used, for example, in mutants in which these genes have lost their function, for example by deletion by conventional methods. In addition, the nucleic acid sequence molecule encoding a selectable marker can be contained in the same vector as the sequence encoding the polypeptide of the present invention or used in the method of the present invention, or introduced into the host cell in a separate vector. Cells that have been stably transfected with the introduced nucleic acid sequence can be identified, for example, by selection (eg, cells incorporating the selectable marker survive while other cells die). The marker gene can be removed or excised from the transgenic cell once it is no longer needed. Techniques for marker gene removal are well known in the art and useful techniques are described above in the "Definitions" section.
本发明还提供了产生相对于对照植物具有增强的非生物胁迫耐受性和/或产量相关性状的转基因植物的方法，包括在植物中引入和表达编码如前文所定义的alfin样多肽或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的任何核酸。The present invention also provides a method for producing transgenic plants with enhanced abiotic stress tolerance and/or yield-related traits relative to control plants, comprising introducing and expressing an alfin-like polypeptide or a YRP polypeptide as defined above in a plant or any nucleic acid of a BRXL polypeptide or a silky-1-like polypeptide or an ARP6 polypeptide or a POP polypeptide or a CRL polypeptide.
更具体地，本发明提供了产生具有增强的非生物胁迫耐受性，特别是增加的(轻度)干旱耐受性的转基因植物的方法，所述方法包括：More specifically, the present invention provides a method for producing transgenic plants with enhanced abiotic stress tolerance, in particular increased (mild) drought tolerance, said method comprising:
(i)向植物或植物细胞中引入和表达编码alfin样多肽或YRP多肽或silky-1样多肽的核酸；和(i) introducing and expressing a nucleic acid encoding an alfin-like polypeptide or a YRP polypeptide or a silky-1-like polypeptide in a plant or a plant cell; and
(ii)在非生物胁迫条件下培养所述植物细胞。(ii) culturing said plant cells under abiotic stress conditions.
(i)中的核酸可以为任何能够编码如本文定义的alfin样多肽或YRP多肽或silky-1样多肽的核酸。The nucleic acid in (i) may be any nucleic acid capable of encoding an alfin-like polypeptide or a YRP polypeptide or a silky-1-like polypeptide as defined herein.
更具体地，本发明还提供了产生相对于对照植物具有增强的产量相关性状的转基因植物的方法，所述方法包括：More specifically, the present invention also provides a method for producing transgenic plants having enhanced yield-related traits relative to control plants, said method comprising:
(i)向植物、植物部分或植物细胞中引入和表达编码BRXL多肽的核酸序列；和(i) introducing and expressing the nucleic acid sequence of coding BRXL polypeptide in plant, plant part or plant cell; With
(ii)在促进植物生长和发育的条件下培养所述植物细胞、植物部分或植物。(ii) culturing said plant cell, plant part or plant under conditions that promote plant growth and development.
(i)中的核酸序列可以为任何能够编码如本文定义的BRXL多肽的核酸序列。The nucleic acid sequence in (i) may be any nucleic acid sequence capable of encoding a BRXL polypeptide as defined herein.
更具体地，本发明还提供了产生具有增强的产量相关性状，特别是增加的种子产量的转基因植物的方法，所述方法包括：More specifically, the present invention also provides a method for producing transgenic plants having enhanced yield-related traits, in particular increased seed yield, said method comprising:
(i)向植物或植物细胞中引入和表达编码ARP6多肽的核酸；和(i) introducing and expressing a nucleic acid encoding an ARP6 polypeptide into a plant or plant cell; and
(ii)在促进植物生长和发育的条件下培养所述植物细胞。(ii) culturing said plant cells under conditions that promote plant growth and development.
(i)中的核酸序列可以为任何能够编码如本文所述的ARP6多肽的核酸。The nucleic acid sequence in (i) may be any nucleic acid capable of encoding an ARP6 polypeptide as described herein.
更具体地，本发明还提供了产生具有增强的产量相关性状，特别是增加的产量的转基因植物的方法，所述方法包括：More specifically, the present invention also provides a method for producing transgenic plants with enhanced yield-related traits, in particular increased yield, said method comprising:
(i)向植物或植物细胞中引入和表达编码POP多肽的核酸；和(i) introducing and expressing a nucleic acid encoding a POP polypeptide into a plant or plant cell; and
(ii)在促进植物生长和发育的条件下培养所述植物细胞。(ii) culturing said plant cells under conditions that promote plant growth and development.
(i)中的核酸序列可以为任何能够编码如本文所述的POP多肽的核酸。The nucleic acid sequence in (i) may be any nucleic acid capable of encoding a POP polypeptide as described herein.
更具体地，本发明还提供了产生具有增强的产量相关性状，特别是增加的(种子)产量和/或收获指数的转基因植物的方法，所述方法包括：More specifically, the present invention also provides a method for producing transgenic plants having enhanced yield-related traits, in particular increased (seed) yield and/or harvest index, said method comprising:
(i)向植物或植物细胞中引入和表达编码CRL多肽的核酸；和(i) introducing and expressing a nucleic acid encoding a CRL polypeptide into a plant or plant cell; and
(ii)在促进植物生长和发育的条件下培养所述植物细胞。(ii) culturing said plant cells under conditions that promote plant growth and development.
(i)中的核酸序列可以为任何能够编码如本文所述的CRL多肽的核酸。The nucleic acid sequence in (i) may be any nucleic acid capable of encoding a CRL polypeptide as described herein.
可以将核酸直接引入植物细胞或植物本身(包括引入植物的组织、器官或任何其它部分)。根据本发明优选的方面，优选通过转化将核酸引入植物。术语“转化”在本文“定义”部分有更详细的说明。Nucleic acids can be introduced directly into plant cells or into the plant itself (including into a tissue, organ or any other part of the plant). According to a preferred aspect of the invention, the nucleic acid is introduced into the plant, preferably by transformation. The term "transformation" is described in more detail in the "Definitions" section herein.
遗传修饰的植物细胞能够通过技术人员熟悉的所有方法再生。合适的方法可见于上述S.D.Kung和R.Wu、Potrykus或者和Willmitzer的出版物。Genetically modified plant cells can be regenerated by all methods familiar to the skilled person. Suitable methods can be found in above mentioned SDKung and R.Wu, Potrykus or and Willmitzer publications.
通常在转化以后，选出存在一个或多个标记的植物细胞或细胞群，所述标记由与目的基因共转移的植物可表达基因编码，接着使转化的材料再生成整个植物。为选择转化的植物，通常将在转化中获得的植物材料置于选择性条件下，从而可将转化的植物与未转化的植物区分开来。例如，可以种植以上述方式获得的种子，并在最初的生长期之后，通过喷雾对其进行合适的选择。另一可能性方案是在使用合适的选择剂的琼脂板上生长种子(酌情在灭菌后)，从而仅转化的种子能够长成植物。可选地，针对可选择标记例如上文所述标记的存在，筛选转化的植物。Typically following transformation, plant cells or populations of cells are selected for the presence of one or more markers encoded by plant-expressible genes co-transferred with the gene of interest, and the transformed material is then regenerated into whole plants. For the selection of transformed plants, the plant material obtained in the transformation is generally subjected to selective conditions, whereby transformed plants can be distinguished from non-transformed plants. For example, the seeds obtained in the manner described above can be planted and, after the initial growth period, subjected to a suitable selection by spraying. Another possibility is to grow the seeds (after sterilization if appropriate) on agar plates with a suitable selection agent so that only transformed seeds are able to grow into plants. Alternatively, transformed plants are screened for the presence of a selectable marker, such as those described above.
DNA转移和再生之后，还可例如用Southern分析(DNA印迹)，评价推定转化的植物，评价目的基因的存在、拷贝数和/或基因组构造。可选的或额外地，可用Northern和/或Western分析(蛋白质印迹)监测新引入的DNA的表达水平，这两种技术都是本领域普通技术人员所公知的。Following DNA transfer and regeneration, putatively transformed plants can also be assessed for the presence, copy number and/or genomic organization of the gene of interest, for example by Southern analysis (Southern blot). Alternatively or additionally, the expression level of the newly introduced DNA can be monitored by Northern and/or Western analysis (Western blot), both techniques are well known to those of ordinary skill in the art.
产生的转化植物可以通过多种方式繁殖，如通过克隆繁殖或经典的育种技术。例如，第一代(或T1)转化的植物可自交，选择纯合的第二代(或T2)转化体，而T2植物可进一步通过经典育种技术繁殖。产生的转化生物体可以呈多种形式。例如，它们可以是转化细胞和非转化细胞的嵌合体；克隆的转化体(例如所有细胞已转化而含有表达盒)；转化的和非转化的组织的嫁接体(例如在植物中，转化的砧木嫁接到非转化的接穗上)。The resulting transformed plants can be propagated in various ways, such as by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants can be selfed, homozygous second generation (or T2) transformants are selected, and the T2 plants can be further propagated by classical breeding techniques. The resulting transformed organism can take a variety of forms. For example, they may be chimeras of transformed and non-transformed cells; clonal transformants (e.g., all cells have been transformed to contain the expression cassette); grafts of transformed and non-transformed tissues (e.g., in plants, transformed rootstock grafted onto non-transformed scions).
本发明显然延及由本文所述任何方法产生的任何植物细胞或植物，以及所有的植物部分及其繁殖体。本发明还延及由任何上述方法产生的原代转化或转染的细胞、组织、器官或整个植物的后代，所述后代的唯一要求是与根据本发明方法所产生的亲本呈现相同的基因型和/或表型特征。The invention expressly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The invention also extends to progeny of primary transformed or transfected cells, tissues, organs or whole plants produced by any of the above methods, the only requirement of which progeny is to exhibit the same genotype as the parent produced according to the method of the invention and/or phenotypic characteristics.
本发明也包括包含编码上文所定义的alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的分离核酸的宿主细胞。根据本发明优选的宿主细胞是植物细胞。对于根据本发明方法所用的核酸或载体、表达盒或构建体或载体，宿主植物原则上有利地为能够合成本发明方法中所用的多肽的所有植物。The invention also includes host cells comprising an isolated nucleic acid encoding an alfin-like polypeptide, or a YRP polypeptide or a BRXL polypeptide or a silky-1-like polypeptide or an ARP6 polypeptide or a POP polypeptide or a CRL polypeptide as defined above. Preferred host cells according to the invention are plant cells. For the nucleic acids or vectors, expression cassettes or constructs or vectors used according to the methods of the invention, host plants are in principle advantageously all plants which are capable of synthesizing the polypeptides used in the methods of the invention.
本发明方法有利地适用于任何植物。尤其可用于本发明方法的植物包括属于植物界超家族的所有植物，尤其是单子叶植物和双子叶植物，包括饲料或牧草豆科植物、观赏植物、粮食作物、乔木或灌木。根据本发明优选的实施方案，植物为作物植物。作物植物的实例包括大豆、向日葵、芸苔、苜蓿、油菜籽、亚麻籽(linseed)、棉花、番茄、马铃薯和烟草。还优选植物是单子叶植物。单子叶植物的实例包括甘蔗。更优选植物是谷类。谷类的实例包括稻、玉米、小麦、大麦、粟、黑麦、黑小麦、高粱、二粒小麦、斯佩耳特小麦(spelt)、黑麦、单粒小麦(einkorn)、埃塞俄比亚画眉草、买罗高粱(milo)和燕麦。The method of the invention is advantageously applicable to any plant. Plants which are particularly useful in the method of the invention include all plants belonging to the superfamily Plantae, especially monocotyledonous and dicotyledonous plants, including forage or pasture legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the invention the plants are crop plants. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, linseed, cotton, tomato, potato and tobacco. It is also preferred that the plants are monocots. Examples of monocots include sugar cane. More preferably the plant is a cereal. Examples of cereals include rice, corn, wheat, barley, millet, rye, triticale, sorghum, emmer, spelt, rye, einkorn, Ethiopian teff, Luo sorghum (milo) and oats.
本发明也延及植物的可收获部分，例如但不限于：种子、叶、果实、花、茎、根、根茎、块茎和球茎，所述可收获部分含有编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的重组核酸。本发明还涉及由这样的植物的可收获部分产生的、优选直接产生的产品，如干丸或粉、油类、脂肪和脂肪酸、淀粉或蛋白质。The invention also extends to harvestable parts of plants, such as but not limited to: seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs, said harvestable parts containing an alfin-like polypeptide, or a YRP polypeptide or BRXL A recombinant nucleic acid of a polypeptide or a silky-1-like polypeptide or an ARP6 polypeptide or a POP polypeptide or a CRL polypeptide. The invention also relates to products, preferably directly produced, such as dry pellets or powders, oils, fats and fatty acids, starches or proteins produced from harvestable parts of such plants.
根据本发明的优选方面，表达的调节是表达的增加。增加核酸或基因或基因产物表达的方法在本领域有充分的文献记载，并且实例在“定义”部分提供。According to a preferred aspect of the invention, the modulation of expression is an increase of expression. Methods of increasing expression of nucleic acids or genes or gene products are well documented in the art and examples are provided in the "Definitions" section.
如上文所述，调节编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸表达的一个优选方法是，在植物中引入和表达编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸；然而，实施所述方法的效果，即增强的非生物胁迫耐受性，也可以利用其他众所周知的技术实现，包括但不限于：T-DNA激活标记、TILLING、同源重组。这些技术的说明在定义部分提供。As mentioned above, a preferred method of modulating the expression of a nucleic acid encoding an alfin-like polypeptide, or a YRP polypeptide, or a BRXL polypeptide, or a silky-1-like polypeptide, or an ARP6 polypeptide, or a POP polypeptide, or a CRL polypeptide, is to introduce and express an alfin-like polypeptide encoding an alfin-like polypeptide in a plant. Polypeptides, or YRP polypeptides or BRXL polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or nucleic acids of CRL polypeptides; however, the effect of implementing the method, i.e. enhanced abiotic stress tolerance, can also be exploited using other well known Technical implementation, including but not limited to: T-DNA activation markers, TILLING, homologous recombination. Descriptions of these techniques are provided in the Definitions section.
本发明还包括在增强植物的任一上述非生物胁迫耐受性中编码如本文所述的alfin样多肽或YRP多肽或silky-1样多肽的核酸的用途，以及这些多肽的用途。The present invention also includes the use of nucleic acids encoding alfin-like polypeptides or YRP polypeptides or silky-1-like polypeptides as described herein, as well as the use of these polypeptides, in enhancing the tolerance of any of the above-mentioned abiotic stresses in plants.
本发明还包括编码如本文所述BRXL多肽的核酸序列的用途，以及这些BRXL多肽的用途：用于在正常生长条件下、在非生物胁迫生长条件(优选渗透胁迫生长条件)下、和在养分可利用率减小的生长条件下、优选在氮可利用率减小的条件下，增强植物的任一上述产量相关性状。The present invention also includes the use of nucleic acid sequences encoding BRXL polypeptides as described herein, and the use of these BRXL polypeptides: under normal growth conditions, under abiotic stress growth conditions (preferably osmotic stress growth conditions), and under nutrient Any of the above yield-related traits are enhanced in plants under growth conditions of reduced availability, preferably under conditions of reduced nitrogen availability.
本发明还包括在增强植物的任一上述产量相关性状中编码如本文所述ARP6多肽的核酸的用途、以及这些ARP6多肽、或POP多肽或CRL多肽的用途。The present invention also includes the use of nucleic acids encoding ARP6 polypeptides as described herein, and the use of these ARP6 polypeptides, or POP polypeptides or CRL polypeptides, in enhancing any of the above yield-related traits in plants.
可以在育种程序中使用编码本文所述alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸、或所述alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽本身，其中鉴定可以与编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的基因遗传连锁的DNA标记。可以使用所述核酸/基因或所述alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽本身定义分子标记。接着可以在育种程序中使用此DNA或蛋白质标记，以选择具有在本发明方法中如上文所定义的增强的非生物胁迫耐受性的植物。Nucleic acids encoding alfin-like polypeptides, or YRP polypeptides or BRXL polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or CRL polypeptides described herein, or said alfin-like polypeptides, or YRP polypeptides or BRXL can be used in breeding programs Polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or CRL polypeptides themselves, wherein identification of genes encoding alfin-like polypeptides, or YRP polypeptides or BRXL polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or CRL polypeptides Genetically linked DNA markers. Molecular markers can be defined using said nucleic acid/gene or said alfin-like polypeptide, or YRP polypeptide or BRXL polypeptide or silky-1-like polypeptide or ARP6 polypeptide or POP polypeptide or CRL polypeptide itself. This DNA or protein marker can then be used in a breeding program to select plants with enhanced abiotic stress tolerance as defined above in the methods of the invention.
编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸/基因的等位基因变体也可以用于标记辅助的育种程序。这类育种程序有时需要使用例如EMS诱变，通过植物诱变处理引入等位基因变异；可选的，此类程序可以起始于一系列无意产生的所谓“天然”起源的等位基因变体。然后通过例如PCR进行等位基因变体的鉴定。随后是选择步骤，用以选择所讨论序列的较好等位基因变体，该变体提供可表现为增加的产量的增强的胁迫耐受性。一般通过监测含有所讨论序列的不同等位基因变体的植物的生长行为来进行选择。可以在温室或田地中监测生长行为。更多任选的步骤包括使经鉴定含有较好等位基因变体的植物与另一植物杂交。例如，可使用这种方法产生感兴趣表型特征的组合。Allelic variants of nucleic acids/genes encoding alfin-like polypeptides, or YRP polypeptides or BRXL polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or CRL polypeptides may also be used in marker-assisted breeding programs. Such breeding programs sometimes require the introduction of allelic variation by mutagenic treatment of plants using, for example, EMS mutagenesis; alternatively, such programs may start with a series of allelic variants of so-called "natural" origin that arise unintentionally . Identification of allelic variants is then performed by, for example, PCR. This is followed by a selection step to select for better allelic variants of the sequence in question which confer enhanced stress tolerance which may manifest as increased yield. Selection is generally performed by monitoring the growth behavior of plants containing different allelic variants of the sequence in question. Growth behavior can be monitored in a greenhouse or in the field. A further optional step involves crossing the plant identified as containing the preferred allelic variant with another plant. For example, combinations of phenotypic features of interest can be generated using this approach.
编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸还可以用作探针对包含其的基因进行遗传和物理作图，以及用作与这些基因连锁的性状的标记。这样的信息可以在植物育种中使用，以培育具有所期望表型的株系。编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸的这类应用仅需要长度至少15个核苷酸的核酸序列。编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸可以用作限制性片段长度多态性(RFLP)标记。可以用编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸探测限制酶切消化的植物基因组DNA的Southern印迹(Sambrook J，Fritsch EF和Maniatis T(1989)《分子克隆：实验室手册》)。随后使用计算机程序如MapMaker(Lander等(1987)Genomics 1：174-181)对产生的带型进行遗传分析，以构建遗传图谱。另外，可以使用所述核酸探测含有一组如下个体的限制性内切酶处理的基因组DNA的Southern印迹，所述该组个体为规定的遗传杂交的亲本和子代。记录DNA多态性的分离，并用于计算编码alfin样多肽、或YRP多肽或BRXL多肽或silky-1样多肽或ARP6多肽或POP多肽或CRL多肽的核酸在先前用此群体所获得的遗传图谱中的位置(Botstein等(1980)Am.J.Hum.Genet.32：314-331)。Nucleic acids encoding alfin-like polypeptides, or YRP polypeptides, or BRXL polypeptides, or silky-1-like polypeptides, or ARP6 polypeptides, or POP polypeptides, or CRL polypeptides can also be used as probes for genetic and physical mapping of genes comprising them, and for use with These genes are linked markers for traits. Such information can be used in plant breeding to develop lines with desired phenotypes. Such use of nucleic acids encoding alfin-like polypeptides, or YRP polypeptides or BRXL polypeptides or silky-1-like polypeptides or ARP6 polypeptides or POP polypeptides or CRL polypeptides requires only nucleic acid sequences of at least 15 nucleotides in length. Nucleic acids encoding alfin-like polypeptides, or YRP polypeptides, or BRXL polypeptides, or silky-1-like polypeptides, or ARP6 polypeptides, or POP polypeptides, or CRL polypeptides can be used as restriction fragment length polymorphism (RFLP) markers. Southern blots of restriction digested plant genomic DNA can be probed with nucleic acids encoding alfin-like polypeptides, or YRP polypeptides, or BRXL polypeptides, or silky-1-like polypeptides, or ARP6 polypeptides, or POP polypeptides, or CRL polypeptides (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual). The resulting banding patterns are then subjected to genetic analysis using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) to construct genetic maps. In addition, the nucleic acid can be used to probe Southern blots of restriction endonuclease-treated genomic DNA containing a set of individuals that are the parents and progeny of a defined genetic cross. Segregation of DNA polymorphisms was recorded and used to calculate nucleic acids encoding alfin-like polypeptides, or YRP polypeptides, or BRXL polypeptides, or silky-1-like polypeptides, or ARP6 polypeptides, or POP polypeptides, or CRL polypeptides in genetic maps previously obtained with this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
有关在遗传作图中使用的植物基因衍生探针的产生和使用，描述于Bematzky和Tanksley(1986)Plant Mol.Biol.Reporter 4：37-41中。众多出版物中描述过用上述方法或其变通形式对特定cDNA克隆进行的遗传作图。例如，可以使用F2杂交群体、回交群体、随机交配群体、近等基因系和其它个体组作图。这类方法是本领域技术人员公知的。The generation and use of plant gene-derived probes for use in genetic mapping is described in Bematzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Genetic mapping of specific cDNA clones using the methods described above or variations thereof are described in numerous publications. For example, F2 cross populations, backcross populations, random mating populations, near isogenic lines, and other groups of individuals can be used for mapping. Such methods are well known to those skilled in the art.
核酸探针也可以用来进行物理作图(即在物理图谱上安置序列；参见Hoheisel等In：Non-mammalian Genomic Analysis：A Practical Guide，Academic press 1996，第319-346页，及其中引用的参考文献)。Nucleic acid probes can also be used for physical mapping (i.e. placing sequences on a physical map; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein. literature).
在另一个实施方案中，核酸探针可用于直接荧光原位杂交(FISH)作图(Trask(1991)Trends Genet.7：149-154)。尽管目前FISH作图的方法倾向使用大的克隆(几个kb到几百个kb；参见Laan等(1995)Genome Res.5：13-20)，但是灵敏性的提高可以允许在FISH作图中应用较短的探针。In another embodiment, nucleic acid probes can be used for direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods for FISH mapping favor the use of large clones (several kb to hundreds of kb; see Laan et al. (1995) Genome Res. 5:13-20), increased sensitivity may allow Use shorter probes.
用于遗传和物理作图的多种基于核酸扩增的方法可以使用所述核酸序列进行。实例包括等位基因特异性扩增(Kazazian(1989)J.Lab.Clin.Med11：95-96)、PCR扩增片段的多态性(CAPS；Sheffield等(1993)Genomics16：325-332)、等位基因特异性连接(Landegren等(1988)Science 241：1077-1080)、核苷酸延伸反应(Sokolov(1990)Nucleic Acid Res.18：3671)、放射杂交作图(Walter等(1997)Nat.Genet.7：22-28)和Happy作图(Dear和Cook(1989)Nucleic Acid Res.17：6795-6807)。为实施这些方法，使用核酸的序列设计和产生用于扩增反应或引物延伸反应的引物对。这类引物的设计是本领域技术人员公知的。在采用基于PCR的遗传作图的方法中，可能需要鉴定作图杂交的亲本之间在相应于本发明核酸序列的区域中的DNA序列差异。然而，这对作图方法通常不是必要的。A variety of nucleic acid amplification-based methods for genetic and physical mapping can be performed using the nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J.Lab.Clin.Med 11:95-96), polymorphism of PCR amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), Allele-specific ligation (Landegren et al. (1988) Science 241: 1077-1080), nucleotide extension reaction (Sokolov (1990) Nucleic Acid Res. 18: 3671), radial hybridization mapping (Walter et al. (1997) Nat . Genet.7: 22-28) and Happy plotting (Dear and Cook (1989) Nucleic Acid Res. 17: 6795-6807). To perform these methods, the sequence of the nucleic acid is used to design and generate primer pairs for amplification reactions or primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be desirable to identify DNA sequence differences between the parents of the mapping cross in regions corresponding to the nucleic acid sequences of the invention. However, this is usually not necessary for graphing methods.
根据本发明方法导致具有如前文所述增强的非生物胁迫耐受性和/或增强的产量相关性状的植物。这些性状还可以组合其它经济上有利的性状，例如其它增强非生物或生物胁迫耐受性的性状、增强的产量相关性状、增强的产量相关性状、对除草剂、杀昆虫剂的耐受性、改变各种构造特征和/或生物化学和/或生理学特征的性状。The method according to the invention leads to plants having enhanced abiotic stress tolerance and/or enhanced yield-related traits as described above. These traits can also be combined with other economically advantageous traits, such as other traits that enhance tolerance to abiotic or biotic stress, enhanced yield-related traits, enhanced yield-related traits, tolerance to herbicides, insecticides, Altering the traits of various structural features and/or biochemical and/or physiological features.
项目project
1.Alfin样多肽1. Alfin-like peptide
1.通过在植物中调节编码alfin样多肽或其直向同源物或旁系同源物的核酸的表达，增强植物的非生物胁迫耐受性的方法。
2.根据项1的方法，其中所述调节的表达通过向植物中引入和表达编码alfin样多肽的核酸来实现。2. The method according to
3.根据项2或3的方法，其中所述编码alfin样多肽的核酸编码表A1所列的任一蛋白质，或是该核酸的部分或能够与该核酸杂交的核酸。3. The method according to item 2 or 3, wherein the nucleic acid encoding an alfin-like polypeptide encodes any protein listed in Table A1, or is a part of the nucleic acid or a nucleic acid capable of hybridizing to the nucleic acid.
4.根据项1至4的任一项的方法，其中所述核酸序列编码表A1中所给出的任何蛋白质的直向同源物或旁系同源物。4. Method according to any one of
5.根据项3或4的任一的方法，其中所述核酸有效地连接至组成型启动子，优选连接至GOS2启动子，最优选连接至来自稻的GOS2启动子。5. Method according to any one of
6.根据项1至5任一项的方法，其中所述编码alfin样多肽的核酸是洋葱(Allium cepa)的核酸。6. The method according to any one of
7.可通过根据项1至6的任一项的方法获得的植物或其部分，包括种子，其中所述植物或其部分包含编码alfin样多肽的重组核酸。7. Plant or part thereof, including seeds, obtainable by a method according to any one of
8.构建体，其包含：8. A construct comprising:
(i)如项1或2中定义的编码alfin样多肽的核酸；(i) a nucleic acid encoding an alfin-like polypeptide as defined in
(ii)能够驱动(a)的核酸序列表达的一个或多个控制序列；和任选地(ii) one or more control sequences capable of driving the expression of the nucleic acid sequence of (a); and optionally
(iii)转录终止序列。(iii) Transcription termination sequence.
9.根据项8的构建体，其中所述控制序列之一是组成型启动子，优选GOS2启动子，最优选来自稻的GOS2启动子。9. Construct according to item 8, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
10.根据项8或9的构建体在用于制备相对于对照植物具有增强的非生物胁迫耐受性的植物的方法中的用途。.10. Use of the construct according to item 8 or 9 in a method for producing plants having increased tolerance to abiotic stress relative to control plants. .
11.利用根据项8或9的构建体转化的植物、植物部分或植物细胞。11. A plant, plant part or plant cell transformed with a construct according to item 8 or 9.
12.用于产生相对于对照植物具有增加的非生物胁迫耐受性的转基因植物的方法，其包括：12. A method for producing a transgenic plant with increased abiotic stress tolerance relative to a control plant, comprising:
(i)向植物中引入和表达编码alfin样多肽的核酸；和(i) introducing and expressing a nucleic acid encoding an alfin-like polypeptide in a plant; and
(ii)在促进非生物胁迫的条件下培养所述植物细胞。(ii) culturing said plant cell under conditions that promote abiotic stress.
13.相对于对照植物具有非生物胁迫耐受性的转基因植物或源自所述转基因植物的转基因植物细胞，其中所述非生物胁迫耐受性因编码alfin样多肽的核酸的被调节的表达而产生。13. A transgenic plant with tolerance to abiotic stress or a transgenic plant cell derived from said transgenic plant with respect to a control plant, wherein said abiotic stress tolerance is due to the regulated expression of a nucleic acid encoding an alfin-like polypeptide produce.
14.根据项7、11或13的转基因植物或源自其的转基因植物细胞，其中所述植物是作物植物或单子叶植物或谷类植物例如稻、玉米、小麦、大麦、粟、黑麦(rye)、黑小麦、高粱、甘蔗、二粒小麦、斯佩耳特小麦、黑麦(secale)、单粒小麦、埃塞俄比亚画眉草、买罗高梁和燕麦。14. The transgenic plant or a transgenic plant cell derived therefrom according to item 7, 11 or 13, wherein said plant is a crop plant or a monocotyledonous plant or a cereal plant such as rice, maize, wheat, barley, millet, rye (rye ), triticale, sorghum, sugar cane, emmer, spelled, rye (secale), einkorn, Ethiopian teff, milo, and oats.
15.根据项14的植物的可收获部分，其中所述可收获部分优选是枝条生物质和/或种子。15. Harvestable parts of a plant according to item 14, wherein said harvestable parts are preferably shoot biomass and/or seeds.
16.从根据项14的植物和/或从根据项15的植物的可收获部分得到的产品。16. Products obtained from plants according to item 14 and/or from harvestable parts of plants according to item 15.
17.编码alfin样多肽的核酸在相对于对照植物增加产量，特别是在增加非生物胁迫耐受性中的用途。17. Use of a nucleic acid encoding an alfin-like polypeptide for increasing yield relative to control plants, in particular for increasing abiotic stress tolerance.
2.YRP多肽2. YRP polypeptide
1.通过在植物中调节编码YRP多肽或其直向同源物或旁系同源物的核酸的表达，增强植物的非生物胁迫耐受性的方法。
2.根据项1的方法，其中所述调节的表达通过向植物中引入和表达编码YRP多肽的核酸来实现。.2. The method according to
3.根据项2或3的方法，其中所述编码YRP多肽的核酸编码表A2所列的任一蛋白质，或是这样的核酸的部分或能够与这样的核酸杂交的核酸。3. The method according to item 2 or 3, wherein the nucleic acid encoding a YRP polypeptide encodes any protein listed in Table A2, or is a part of such a nucleic acid or a nucleic acid capable of hybridizing to such a nucleic acid.
4.根据项1至4的任一项的方法，其中所述核酸序列编码表A2中所给出的任何蛋白质的直向同源物或旁系同源物。4. Method according to any one of
5.根据项3或4的任一的方法，其中所述核酸有效地连接至组成型启动子，优选连接至GOS2启动子，最优选连接至来自稻的GOS2启动子。5. Method according to any one of
6.根据项1至5任一项的方法，其中所述编码YRP多肽的核酸是大麦的核酸。6. The method according to any one of
7.可通过根据项1至6的任一项的方法获得的植物或其部分，包括种子，其中所述植物或其部分包含编码YRP多肽的重组核酸。7. Plant or part thereof, including seeds, obtainable by a method according to any one of
8.构建体，其包含：8. A construct comprising:
(i)如项1或2中定义的编码YRP多肽的核酸；(i) a nucleic acid encoding a YRP polypeptide as defined in
(ii)能够驱动(a)的核酸序列表达的一个或多个控制序列；和任选地(ii) one or more control sequences capable of driving the expression of the nucleic acid sequence of (a); and optionally
(iii)转录终止序列。(iii) Transcription termination sequence.
9.根据项8的构建体，其中所述控制序列之一是组成型启动子，优选GOS2启动子，最优选来自稻的GOS2启动子。9. Construct according to item 8, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
10.根据项8或9的构建体在用于制备相对于对照植物具有增强的非生物胁迫耐受性的植物的方法中的用途。10. Use of the construct according to item 8 or 9 in a method for producing plants having increased tolerance to abiotic stress relative to control plants.
11.利用根据项8或9的构建体转化的植物、植物部分或植物细胞。11. A plant, plant part or plant cell transformed with a construct according to item 8 or 9.
12.用于产生相对于对照植物具有增加的非生物胁迫耐受性的转基因植物的方法，其包括：12. A method for producing a transgenic plant with increased abiotic stress tolerance relative to a control plant, comprising:
(i)向植物中引入和表达编码YRP多肽的核酸；和(i) introducing and expressing a nucleic acid encoding a YRP polypeptide in a plant; and
(ii)在促进非生物胁迫的条件下培养所述植物细胞。(ii) culturing said plant cell under conditions that promote abiotic stress.
13.相对于对照植物具有非生物胁迫耐受性的转基因植物细胞或源自所述转基因植物的转基因植物细胞，其中所述非生物胁迫耐受性因编码YRP多肽的核酸的被调节的表达而产生。13. have the transgenic plant cell of abiotic stress tolerance with respect to control plant or be derived from the transgenic plant cell of described transgenic plant, wherein said abiotic stress tolerance is due to the regulated expression of the nucleic acid of coding YRP polypeptide produce.
14.根据项7、11或13的转基因植物或源自其的转基因植物细胞，其中所述植物是作物植物或单子叶植物或谷类植物例如稻、玉米、小麦、大麦、粟、黑麦、黑小麦、高粱、甘蔗、二粒小麦、斯佩耳特小麦、黑麦、单粒小麦、埃塞俄比亚画眉草、买罗高梁和燕麦。14. The transgenic plant or a transgenic plant cell derived therefrom according to item 7, 11 or 13, wherein said plant is a crop plant or a monocotyledonous plant or a cereal plant such as rice, maize, wheat, barley, millet, rye, rye Wheat, Sorghum, Sugar Cane, Emmer, Spelled, Rye, Einkorn, Ethiopian Teff, Milo, and Oats.
15.根据项14的植物的可收获部分，其中所述可收获部分优选是枝条生物质和/或种子。15. Harvestable parts of a plant according to item 14, wherein said harvestable parts are preferably shoot biomass and/or seeds.
16.从根据项14的植物和/或从根据项15的植物的可收获部分得到的产品。16. Products obtained from plants according to item 14 and/or from harvestable parts of plants according to item 15.
17.编码YRP多肽的核酸在相对于对照植物增加产量，特别是在增加非生物胁迫耐受性中的用途。17. Use of a nucleic acid encoding a YRP polypeptide for increasing yield relative to control plants, in particular for increasing tolerance to abiotic stress.
3.Brevis Radix样(BRXL)多肽3. Brevis Radix-like (BRXL) polypeptide
1)相对于对照植物增强植物的产量相关性状的方法，其包括增加编码Brevis Radix样(BRXL)多肽的核酸序列在植物中的表达和任选地选择具有增强的产量相关性状的植物，其中所述BRXL多肽包含至少2个具有InterPro登录号IPR013591DZC结构域(PFAM登录号PF08381 DZC)的BRX结构域。1) A method for enhancing the yield-related traits of plants with respect to control plants, which comprises increasing the expression of a nucleic acid sequence encoding aBrevisRadix- like (BRXL) polypeptide in a plant and optionally selecting plants with enhanced yield-related traits , wherein the BRXL polypeptide comprises at least two BRX domains having InterPro accession number IPR013591DZC domain (PFAM accession number PF08381 DZC).
2)根据项1的方法，其中所述BRXL多肽包含(i)按照递增的优选次序与如SEQ ID NO：65所示的BRX结构域至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性；和(ii)按照递增的优选次序与如SEQ IDNO：82所示的BRX结构域至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。2) The method according to
3)根据项1或2的方法，其中所述BRXL多肽包含(i)按照递增的优选次序与如SEQ ID NO：83所示的保守结构域1(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性；和(ii)按照递增的优选次序与如SEQ ID NO：84所示的保守结构域2(包含BRX结构域)至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。3) The method according to
4)根据项3的方法，其中所述BRXL多肽包含(i)按照递增的优选次序与如SEQ ID NO：85所示的保守结构域3至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性；和(ii)按照递增的优选次序与如SEQ ID NO：86所示的保守结构域4至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。4) The method according to item 3, wherein the BRXL polypeptide comprises (i) at least 50%, 55%, 60%, 65%, 70% of the conserved domain 3 shown in SEQ ID NO: 85 in an increasing preferred order %, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher amino acid sequence identity; and (ii) in order of increasing preference with those shown in SEQ ID NO: 86 The conserved
5)根据任何前述项的方法，其中所述BRXL多肽按照递增的优选次序与如SEQ ID NO：18所示的多肽或与本文中表A所给出的任何多肽序列具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、98％、99％或更高的氨基酸序列同一性。5) The method according to any of the preceding items, wherein the BRXL polypeptide has at least 40%, 45% with the polypeptide shown in SEQ ID NO: 18 or with any polypeptide sequence given in Table A herein in an increasing preferred order , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher amino acid sequence identity.
6)根据任何前述项的方法，其中在酵母双杂交测定中所述BRXL多肽与其本身或与另一种BRLX多肽相互作用。6) The method according to any preceding item, wherein said BRXL polypeptide interacts with itself or with another BRLX polypeptide in a yeast two-hybrid assay.
7)根据任何前述项的方法，其中所述编码BRXL多肽的核酸序列由表A3所给出的任一SEQ ID NO核酸序列表示、或是其部分、或是能够与表A3所给出的任意一个核酸序列或与其互补序列杂交的序列。7) The method according to any preceding item, wherein the nucleic acid sequence encoding the BRXL polypeptide is represented by any SEQ ID NO nucleic acid sequence given in Table A3, or a part thereof, or any combination that can be given in Table A3 A nucleic acid sequence or a sequence that hybridizes to its complement.
8)根据任何前述项的方法，其中所述核酸序列编码表A3所给出的任何SEQ ID NO多肽序列的直向同源物或旁系同源物。8) The method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or a paralogue of any SEQ ID NO polypeptide sequence given in Table A3.
9)根据任何前述项的方法，其中所述增加的表达通过T-DNA激活标记、TILLING、或同源重组中的任一个或多个来实现。9) The method according to any preceding item, wherein said increased expression is achieved by any one or more of T-DNA activation tagging, TILLING, or homologous recombination.
10)根据任何前述项的方法，其中所述增加的表达通过向植物中引入和表达编码BRXL多肽的核酸序列来实现。10) The method according to any preceding item, wherein said increased expression is achieved by introducing and expressing into a plant a nucleic acid sequence encoding a BRXL polypeptide.
11)根据任何前述项的方法，其中述增加的产量相关性状是如下一种或多种：增加的植物高度和增加的千粒重(TKW)。11) The method according to any preceding item, wherein said increased yield-related traits are one or more of: increased plant height and increased thousand kernel weight (TKW).
12)根据任何前述项的方法，其中所述核酸序列有效地连接至组成型启动子。12) The method according to any preceding item, wherein said nucleic acid sequence is operably linked to a constitutive promoter.
13)根据项12的方法，其中所述组成型启动子是GOS2启动子，优选来自稻的GOS2启动子，最优选如SEQ ID NO：87所示的GOS2启动子。13) The method according to item 12, wherein the constitutive promoter is a GOS2 promoter, preferably a GOS2 promoter from rice, most preferably a GOS2 promoter as shown in SEQ ID NO: 87.
14)据项任何前述项的方法，其中所述编码BRXL多肽的核酸序列来自植物，更优选来自双子叶植物，更优选来自杨柳科，最优选核酸序列来自毛果杨。14) The method according to any preceding item, wherein the nucleic acid sequence encoding a BRXL polypeptide is from a plant, more preferably from a dicotyledonous plant, more preferably from Salicaceae, most preferably from Populus trichocarpa.
15)可通过任何前述项的方法获得的植物或其部分(包括种子)或植物细胞，其中所述植物、其部分或细胞包含编码BRXL多肽的分离的核酸转基因。15) Plants or parts thereof (including seeds) or plant cells obtainable by the method of any preceding item, wherein said plants, parts or cells thereof comprise an isolated nucleic acid transgene encoding a BRXL polypeptide.
16)分离的核酸序列，其选自：16) An isolated nucleic acid sequence selected from the group consisting of:
(i)如SEQ ID NO：75、SEQ ID NO：77或SEQ ID NO：79的任一个所示的核酸序列；(i) a nucleic acid sequence as shown in any one of SEQ ID NO:75, SEQ ID NO:77 or SEQ ID NO:79;
(ii)如SEQ ID NO：75、SEQ ID NO：77或SEQ ID NO：79的任一个所示的核酸序列的互补序列；(ii) as SEQ ID NO:75, SEQ ID NO:77 or the complementary sequence of the nucleic acid sequence shown in any one of SEQ ID NO:79;
(iii)编码BRXL多肽的核酸序列，其中所述多肽按照递增的优选次序与SEQ ID NO：76、SEQ ID NO：78或SEQ ID NO：80的任一个所示的多肽序列具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的氨基酸序列同一性。.(iii) a nucleic acid sequence encoding a BRXL polypeptide, wherein said polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity. .
17)分离的多肽序列，其选自：17) An isolated polypeptide sequence selected from the group consisting of:
(i)如SEQ ID NO：76、SEQ ID NO：78或SEQ ID NO：80的任一个所示的多肽序列；(i) a polypeptide sequence as shown in any one of SEQ ID NO:76, SEQ ID NO:78 or SEQ ID NO:80;
(ii)按照递增的优选次序与如SEQ ID NO：76、SEQ ID NO：78或SEQ ID NO：80的任一个所示的多肽序列具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的氨基酸序列同一性的多肽序列；(ii) have at least 50%, 55%, 60%, 65%, 70% of the polypeptide sequence shown in any one of SEQ ID NO: 76, SEQ ID NO: 78 or SEQ ID NO: 80 in increasing order of preference %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher amino acid sequence identity of the polypeptide sequence;
(iii)上述(i)或(ii)所给出的任何多肽序列的衍生物。(iii) Derivatives of any of the polypeptide sequences given in (i) or (ii) above.
18)构建体，其包含：18) A construct comprising:
(a)如项1至8或16之任一中定义的编码BRXL多肽的核酸序列；(a) nucleotide sequence as defined in any one of
(b)能够驱动(a)的核酸序列表达的一个或多个控制序列；和任选地(b) one or more control sequences capable of driving the expression of the nucleic acid sequence of (a); and optionally
(c)转录终止序列。.(c) Transcription termination sequence. .
19)根据项18的构建体，其中所述控制序列是组成型启动子。19) Construct according to item 18, wherein said control sequence is a constitutive promoter.
20)根据项19的构建体，其中所述组成型启动子是GOS2启动子，优选稻的GOS2启动子，最优选如SEQ ID NO：87所示的GOS2启动子。20) The construct according to item 19, wherein the constitutive promoter is a GOS2 promoter, preferably the GOS2 promoter of rice, most preferably the GOS2 promoter shown in SEQ ID NO: 87.
21)根据项18至20之任一的构建体在用于产生相对于对照植物具有增强的产量相关性状的植物的方法中的用途，所述增强的产量相关性状是如下一种或多种：增加的植物高度、每株植物增加的种子产量、增加的饱满种子数和增加的千粒重(TKW)。21) Use of a construct according to any one of items 18 to 20 in a method for producing plants having enhanced yield-related traits relative to control plants, said enhanced yield-related traits being one or more of the following: Increased plant height, increased seed yield per plant, increased number of filled seeds and increased thousand kernel weight (TKW).
22)利用项18至20的任一项的构建体转化的植物、植物部分或植物细胞。22) A plant, plant part or plant cell transformed with the construct of any one of items 18 to 20.
23)用于产生相对于对照植物具有增强的产量性状的转基因植物的方法，其包括：23) A method for producing transgenic plants with enhanced yield traits relative to control plants, comprising:
(i)向植物、植物部分或植物细胞中引入和表达如项1至8或16的任一项中定义的编码BRXL多肽的核酸序列；和(i) introduce and express in plant, plant part or plant cell the nucleic acid sequence of coding BRXL polypeptide as defined in any one of
(ii)在促进植物生长和发育的条件下培养所述植物细胞、植物部分或植物。(ii) culturing said plant cell, plant part or plant under conditions that promote plant growth and development.
24)相对于对照植物具有增强的产量相关性状的转基因植物，或源自所述转基因植物的转基因植物细胞，其中所述增强的产量相关性状因项1至8或16的任一项中定义的编码BRXL多肽的分离核酸序列的表达增加而产生。24) A transgenic plant having enhanced yield-related traits relative to control plants, or a transgenic plant cell derived from said transgenic plant, wherein said enhanced yield-related traits are as defined in any one of
25)根据项14、22或24的转基因植物或源自其的转基因植物细胞，其中所述植物是作物植物或单子叶植物或谷类植物例如稻、玉米、小麦、大麦、粟、黑麦、黑小麦、高粱和燕麦。25) A transgenic plant or a transgenic plant cell derived therefrom according to
26)根据项25的植物的可收获部分，所述可收获部分包含编码BRXL多肽的分离的核酸序列，其中所述可收获部分优选是种子。26) Harvestable parts of a plant according to
27)从根据项25的植物和/或从根据项26的植物的可收获部分产生的产品。27) Products produced from a plant according to
28)如项1至8或16的任一项中定义的编码BRXL多肽的核酸序列在增加产量相关性状中的用途，所述产量相关性状包括如下一种或多种：增加的植物高度和增加的千粒重(TKW)。28) Use of the nucleic acid sequence encoding a BRXL polypeptide as defined in any one of
4.silky1样多肽4. silky1-like polypeptide
1.通过在植物中调节编码silky-1样多肽或其直向同源物或旁系同源物的核酸的表达，增强植物的非生物胁迫耐受性的方法。
2.根据项1的方法，其中所述调节的表达通过向植物中引入和表达编码silky-1样多肽的核酸来实现。2. The method according to
3.根据项2或3的方法，其中所述编码silky-1样多肽的核酸编码表A4所列的任一蛋白质，或是这样的核酸的部分或能够与这样的核酸杂交的核酸。3. The method according to item 2 or 3, wherein the nucleic acid encoding a silky-1-like polypeptide encodes any of the proteins listed in Table A4, or is a part of such a nucleic acid or a nucleic acid capable of hybridizing to such a nucleic acid.
4.根据项1至4的任一项的方法，其中所述核酸序列编码表A4中所给出的任何蛋白质的直向同源物或旁系同源物。4. Method according to any one of
5.根据项3或4的任一的方法，其中所述核酸有效地连接至组成型启动子，优选连接至GOS2启动子，最优选连接至来自稻的GOS2启动子。5. Method according to any one of
6.根据项1至5任一项的方法，其中所述编码silky-1样多肽的核酸是毛果杨的核酸。6. The method according to any one of
7.可通过根据项1至6的任一项的方法获得的植物或其部分，包括种子，其中所述植物或其部分包含编码silky-1样多肽的重组核酸7. A plant or part thereof, including seeds, obtainable by a method according to any one of
8.构建体，其包含：8. A construct comprising:
(i)如项1或2中定义的编码silky-1样多肽的核酸；(i) a nucleic acid encoding a silky-1 sample polypeptide as defined in
(ii)能够驱动(a)的核酸序列表达的一个或多个控制序列；和任选地(ii) one or more control sequences capable of driving the expression of the nucleic acid sequence of (a); and optionally
(iii)转录终止序列。(iii) Transcription termination sequence.
9.根据项8的构建体，其中所述控制序列之一是组成型启动子，优选GOS2启动子，最优选来自稻的GOS2启动子。9. Construct according to item 8, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
10.根据项8或9的构建体在用于制备相对于对照植物具有增强的非生物胁迫耐受性的植物的方法中的用途。10. Use of the construct according to item 8 or 9 in a method for producing plants having increased tolerance to abiotic stress relative to control plants.
11.利用根据项8或9的构建体转化的植物、植物部分或植物细胞。11. A plant, plant part or plant cell transformed with a construct according to item 8 or 9.
12.用于产生相对于对照植物具有增加的非生物胁迫耐受性的转基因植物的方法，其包括：12. A method for producing a transgenic plant with increased abiotic stress tolerance relative to a control plant, comprising:
(i)向植物中引入和表达编码silky-1样多肽的核酸；和(i) introducing and expressing a nucleic acid encoding a silky-1-like polypeptide into a plant; and
(ii)在促进非生物胁迫的条件下培养所述植物细胞。(ii) culturing said plant cell under conditions that promote abiotic stress.
13.相对于对照植物具有非生物胁迫耐受性的转基因植物细胞或源自所述转基因植物的转基因植物细胞，其中所述非生物胁迫耐受性因编码ilky-1样多肽的核酸的被调节的表达而产生。13. A transgenic plant cell having tolerance to abiotic stress or a transgenic plant cell derived from said transgenic plant relative to a control plant, wherein said tolerance to abiotic stress is regulated by a nucleic acid encoding an ilky-1-like polypeptide produced by the expression.
14.根据项7、11或13的转基因植物或源自其的转基因植物细胞，其中所述植物是作物植物或单子叶植物或谷类植物例如稻、玉米、小麦、大麦、粟、黑麦、黑小麦、高粱、甘蔗、二粒小麦、斯佩耳特小麦、黑麦、单粒小麦、埃塞俄比亚画眉草、买罗高梁和燕麦。14. The transgenic plant or a transgenic plant cell derived therefrom according to item 7, 11 or 13, wherein said plant is a crop plant or a monocotyledonous plant or a cereal plant such as rice, maize, wheat, barley, millet, rye, rye Wheat, Sorghum, Sugar Cane, Emmer, Spelled, Rye, Einkorn, Ethiopian Teff, Milo, and Oats.
15.根据项14的植物的可收获部分，其中所述可收获部分优选是枝条生物质和/或种子。15. Harvestable parts of a plant according to item 14, wherein said harvestable parts are preferably shoot biomass and/or seeds.
16.从根据项14的植物和/或从根据项15的植物的可收获部分得到的产品。16. Products obtained from plants according to item 14 and/or from harvestable parts of plants according to item 15.
17.编码silky-1样多肽的核酸在相对于对照植物增加产量，特别是在增加非生物胁迫耐受性中的用途。17. Use of a nucleic acid encoding a silky-1-like polypeptide for increasing yield relative to control plants, in particular for increasing abiotic stress tolerance.
5.ARP6多肽5. ARP6 polypeptide
1.用于相对于对照植物在植物中增强产量相关性状的方法，其包括在植物中调节编码ARP6多肽的核酸的表达。
2.根据项1的方法，其中所述ARP6多肽按照递增的优选次序与如SEQ ID NO：102、SEQ ID NO：104、SEQ ID NO：106、SEQ ID NO：108、SEQ ID NO：110和SEQ ID NO：112的任一个所示的氨基酸具有至少30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的全序列同一性。2. The method according to item 1, wherein the ARP6 polypeptide is in an increasing preferred order with such as SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110 and Any one of the amino acids shown in SEQ ID NO: 112 has at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% , 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity.
3.根据项1或2的方法，其中所述调节的表达通过向植物中引入和表达编码ARP6多肽的核酸来实现。3. Method according to
4.根据项1至3的任一项的方法，其中所述编码ARP6多肽的核酸编码表A5所列的任一蛋白质，或是这样的核酸的部分或能够与这样的核酸杂交的核酸。4. The method according to any one of
5.根据项1至4的任一项的方法，其中所述核酸序列编码表A5中所给出的任何蛋白质的直向同源物或旁系同源物。5. Method according to any one of
6.根据任何前述项的方法，其中述增加的产量相关性状包括，相对于对照植物，增加的产量，优选增加的生物量和/或增加的种子产量。.6. Method according to any preceding item, wherein said increased yield-related traits comprise increased yield, preferably increased biomass and/or increased seed yield, relative to control plants. .
7.根据项1至6的任一项的方法，其中所述增强的产量相关性状在非胁迫条件下获得。7. Method according to any one of
8.根据项1至6的任一项的方法，其中所述增强的产量相关性状在干旱胁迫、盐胁迫或氮缺乏的条件下获得。8. Method according to any one of
9.根据项3至8的任一项的方法，其中所述核酸有效地连接至组成型启动子，优选连接至GOS2启动子，最优选连接至来自稻的GOS2启动子。9. Method according to any one of items 3 to 8, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
10.权利要求1至9的任一项的方法，其中所述编码ARP6多肽的核酸是植物来源的，优选来自双子叶植物，优选来自十字花科，更优选来自拟南芥属，最优选来自拟南芥。10. The method of any one of
11.可通过项1至10的任一项的方法获得的植物或其部分，包括种子，其中所述植物或其部分包含编码ARP6多肽的重组核酸11. Plant or part thereof, including seeds, obtainable by the method of any one of
12.构建体，其包含：12. A construct comprising:
(ii)如项1或2中定义的编码ARP6多肽的核酸；(ii) a nucleic acid encoding an ARP6 polypeptide as defined in
(iii)能够驱动(a)的核酸序列表达的一个或多个控制序列；和任选地(iii) one or more control sequences capable of driving the expression of the nucleic acid sequence of (a); and optionally
(iv)转录终止序列。(iv) Transcription termination sequence.
13.根据项12的构建体，其中所述控制序列之一是组成型启动子，优选GOS2启动子，最优选来自稻的GOS2启动子。13. Construct according to item 12, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
14.根据项12或13的构建体在用于制备相对于对照植物具有增加的产量，特别是增加的生物量和/或增加的种子产量的植物的方法中的用途。14. Use of the construct according to item 12 or 13 in a method for producing plants having increased yield, in particular increased biomass and/or increased seed yield relative to control plants.
15.利用根据项12或13的构建体转化的植物、植物部分或植物细胞。15. A plant, plant part or plant cell transformed with a construct according to item 12 or 13.
16.用于产生相对于对照植物具有增加的产量，特别是增加的生物量和/或增加的种子产量的转基因植物的方法，其包括：16. A method for producing a transgenic plant with increased yield, particularly increased biomass and/or increased seed production relative to control plants, comprising:
(i)向植物中引入和表达如项1或2中定义的编码ARP6多肽的核酸；和(i) introducing and expressing a nucleic acid encoding an ARP6 polypeptide as defined in
(ii)在促进植物生长和发育的条件下培养所述植物细胞。(ii) culturing said plant cells under conditions that promote plant growth and development.
17.相对于对照植物具有增加的产量，特别是增加的生物量和/或增加的种子产量的转基因植物、或源自所述转基因植物的转基因植物细胞，其中所述增加的产量因如项1或2中定义的编码ARP6多肽的核酸的被调节的表达而产生。17. A transgenic plant with increased yield, particularly increased biomass and/or increased seed yield, or a transgenic plant cell derived from said transgenic plant, with respect to a control plant, wherein said increased yield is due to or the regulated expression of the nucleic acid encoding the ARP6 polypeptide defined in 2.
18.根据项11、15或17的转基因植物或源自其的转基因植物细胞，其中所述植物是作物植物或单子叶植物或谷类植物例如稻、玉米、小麦、大麦、粟、黑麦、黑小麦、高粱、二粒小麦、斯佩耳特小麦、黑麦、单粒小麦、埃塞俄比亚画眉草、买罗高梁和燕麦。18. The transgenic plant or a transgenic plant cell derived therefrom according to item 11, 15 or 17, wherein said plant is a crop plant or a monocotyledonous plant or a cereal plant such as rice, maize, wheat, barley, millet, rye, rye Wheat, Sorghum, Emmer, Spelt, Rye, Einkorn, Ethiopian Teff, Milo, and Oats.
19.根据项18的植物的可收获部分，其中所述可收获部分优选是枝条生物质和/或种子。19. Harvestable parts of a plant according to item 18, wherein said harvestable parts are preferably shoot biomass and/or seeds.
20.从根据项18的植物和/或从根据项19的植物的可收获部分得到的产品。20. Products obtained from plants according to item 18 and/or from harvestable parts of plants according to item 19.
21.编码ARP6多肽的核酸在相对于对照植物增加植物的产量，特别是在增加种子产量和/或枝条生物量中的用途。21. Use of a nucleic acid encoding an ARP6 polypeptide for increasing the yield of a plant relative to control plants, in particular for increasing seed yield and/or shoot biomass.
6.POP多肽6. POP polypeptide
1.相对于对照植物增强植物的产量相关性状的方法，其包括调节编码脯氨酰寡肽酶(POP)多肽的核酸在植物中的表达，其中所述POP多肽包含肽酶S9结构域(Pfam登录号PF00326)和优选还包括DPPIV_N结构域(Pfam登录号PF00930)。1. A method for enhancing the yield-related traits of plants with respect to control plants, comprising regulating the expression in plants of a nucleic acid encoding a prolyl oligopeptidase (POP) polypeptide, wherein the POP polypeptide comprises a peptidase S9 domain (Pfam accession number PF00326) and preferably also includes the DPPIV_N domain (Pfam accession number PF00930).
2.根据项1的方法，其中所述POP多肽包含基序1至14(SEQ IDNO：118至SEQ ID NO：131)中的一个或多个。2. The method according to
3.根据项1或2的方法，其中所述调节的表达通过向植物中引入和表达编码POP多肽的核酸来实现。3. The method according to
4.根据项1至3的任一项的方法，其中所述编码POP多肽的核酸编码表A6所列的任一蛋白质，或是这样的核酸的部分或能够与这样的核酸杂交的核酸。4. The method according to any one of
5.根据项1至4的任一项的方法，其中所述核酸序列编码表A6中所给出的任何蛋白质的直向同源物或旁系同源物。5. Method according to any one of
6.根据任何前述项的方法，其中所述增强的产量相关性状包括，相对于对照植物，增加的产量，优选增加的生物量和/或增加的种子产量，和/或改变的开花时间。6. Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably increased biomass and/or increased seed production, and/or altered flowering time relative to control plants.
7.根据项1至6的任一项的方法，其中所述增强的产量相关性状在非胁迫条件下获得。7. Method according to any one of
8.根据项3至7的任一项的方法，其中所述核酸有效地连接至组成型启动子，优选连接至GOS2启动子，最优选连接至来自稻的GOS2启动子。8. Method according to any one of items 3 to 7, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
9.权利要求1至8的任一项的方法，其中所述编码POP多肽的核酸是植物来源的，优选来自双子叶植物，优选来自十字花科，更优选来自拟南芥属，最优选来自拟南芥。9. The method of any one of
10.可通过项1至9的任一项的方法获得的植物或其部分，包括种子，其中所述植物或其部分包含编码POP多肽的重组核酸。10. Plant or part thereof, including seeds, obtainable by the method of any one of
11.构建体，其包含：11. A construct comprising:
(i)如项1或2中定义的编码POP多肽的核酸；(i) a nucleic acid encoding a POP polypeptide as defined in
(ii)能够驱动(a)的核酸序列表达的一个或多个控制序列；和任选地(ii) one or more control sequences capable of driving the expression of the nucleic acid sequence of (a); and optionally
(iii)转录终止序列。(iii) Transcription termination sequence.
12.根据项11的构建体，其中所述控制序列之一是组成型启动子，优选GOS2启动子，最优选来自稻的GOS2启动子。12. Construct according to item 11, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
13.根据项11或12的构建体在用于制备相对于对照植物具有增加的产量，特别是增加的生物量和/或增加的种子产量的植物的方法中的用途。13. Use of a construct according to item 11 or 12 in a method for producing plants having increased yield, in particular increased biomass and/or increased seed yield relative to control plants.
14.根据项11或12的构建体在用于制备相对于对照植物具有改变的开花时间的植物的方法中的用途。14. Use of a construct according to item 11 or 12 in a method for producing plants having an altered flowering time relative to control plants.
15.利用根据项11或12的构建体转化的植物、植物部分或植物细胞。15. A plant, plant part or plant cell transformed with a construct according to item 11 or 12.
16.用于产生相对于对照植物具有增加的产量和/或改变的开花时间，特别是增加的生物量和/或增加的种子产量，的转基因植物的方法，其包括：16. A method for producing transgenic plants with increased yield and/or altered flowering time, particularly increased biomass and/or increased seed production, relative to control plants, comprising:
(i)向植物中引入和表达如项1或2中定义的编码POP多肽的核酸；和(i) introducing and expressing a nucleic acid encoding a POP polypeptide as defined in
(ii)在促进植物生长和发育的条件下培养所述植物细胞。.(ii) culturing said plant cells under conditions that promote plant growth and development. .
17.相对于对照植物具有增加的产量，特别是增加的生物量和/或增加的种子产量的转基因植物、或源自所述转基因植物的转基因植物细胞，其中所述增加的产量因如项1或2中定义的编码POP多肽的核酸的被调节的表达而产生。17. A transgenic plant with increased yield, particularly increased biomass and/or increased seed yield, or a transgenic plant cell derived from said transgenic plant, with respect to a control plant, wherein said increased yield is due to or the regulated expression of the nucleic acid encoding the POP polypeptide defined in 2.
18.根据项10、15或17的转基因植物或源自其的转基因植物细胞，其中所述植物是作物植物或单子叶植物或谷类植物例如稻、玉米、小麦、大麦、粟、黑麦、黑小麦、高粱、二粒小麦、斯佩耳特小麦、黑麦、单粒小麦、埃塞俄比亚画眉草、买罗高梁和燕麦。18. The transgenic plant or a transgenic plant cell derived therefrom according to
19.根据项18的植物的可收获部分，其中所述可收获部分优选是枝条生物质和/或种子。19. Harvestable parts of a plant according to item 18, wherein said harvestable parts are preferably shoot biomass and/or seeds.
20.从根据项18的植物和/或从根据项18的植物的可收获部分得到的产品。20. Products obtained from plants according to item 18 and/or from harvestable parts of plants according to item 18.
21.编码POP多肽的核酸在相对于对照植物增加植物的产量，特别是在增加种子产量和/或枝条生物量中的用途。21. Use of a nucleic acid encoding a POP polypeptide for increasing the yield of a plant relative to control plants, in particular for increasing seed yield and/or shoot biomass.
22.编码POP多肽的核酸在相对于对照植物改变植物的开花时间中的用途。22. Use of a nucleic acid encoding a POP polypeptide for altering the flowering time of a plant relative to a control plant.
7.CRL多肽7. CRL polypeptide
1.用于相对于对照植物在植物中增强产量相关性状的方法，其包括在植物中调节编码CRL多肽的核酸的表达，其中所述CRL多肽包含DUF206结构域。
2.根据项1的方法，其中所述CRL多肽包含按照递增的优选次序与如下任一个或多个基序具有至少50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性的基序：2. The method according to
(i)基序15：EQAFWRxPXKPFRQR(SEQ ID NO：207)；(i) Motif 15: EQAFWRxPXKPFRQR (SEQ ID NO: 207);
(ii)基序16：NFCDR(SEQ ID NO：208)；(ii) Motif 16: NFCDR (SEQ ID NO: 208);
(iii)基序17：RGKRCLYEGS(SEQ ID NO：209)；(iii) Motif 17: RGKRCLYEGS (SEQ ID NO: 209);
(iv)基序18：QVWGxKXGPYEFK(SEQ ID NO：210)；(iv) Motif 18: QVWGxKXGPYEFK (SEQ ID NO: 210);
其中X代表任意氨基酸。wherein X represents any amino acid.
3.根据项1或2的方法，其中所述调节的表达通过向植物中引入和表达编码CRL多肽的核酸来实现。3. The method according to
4.根据项1至3的任一项的方法，其中所述编码CRL多肽的核酸编码表A7中所列的任一蛋白质，或是这样的核酸的部分，或是能够与这样的核酸杂交的核酸。4. The method according to any one of
5.根据项1至4的任一项的方法，其中所述核酸序列编码表A7所给出的任何蛋白质的直向同源物或旁系同源物。5. Method according to any one of
6.根据任何前述项的方法，其中所述增强的产量相关性状包括，相对于对照植物，增加的产量，优选增加的生物量和/或增加的种子产量。6. Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably increased biomass and/or increased seed yield, relative to control plants.
7.根据项1至6的任一项的方法，其中在非胁迫条件下获得所述增强的产量相关性状。7. Method according to any one of
8.根据项1至6的任一项的方法，其中在干旱胁迫、盐胁迫或氮缺乏的条件下获得所述增强的产量相关性状。8. Method according to any one of
9.根据项3至8的任一项的方法，其中所述核酸有效地连接至组成型启动子，优选GOS2启动子，更优选来自稻的GOS2启动子。9. Method according to any one of items 3 to 8, wherein said nucleic acid is operably linked to a constitutive promoter, preferably a GOS2 promoter, more preferably a GOS2 promoter from rice.
10.根据项1至9的任一项的方法，其中所述编码CRL多肽的核酸是植物来源的，优选来自双子叶植物，更优选来自十字花科，更优选来自拟南芥属，最优选来自拟南芥。10. The method according to any one of
11.可通过项1至10的任一项的方法获得的植物或其部分，包括种子，其中所述植物或其部分包含编码CRL多肽的重组核酸。11. Plant or part thereof, including seeds, obtainable by the method of any one of
12.构建体，其包含：12. A construct comprising:
(i)如项1或2中定义的编码CRL多肽的核酸；(i) a nucleic acid encoding a CRL polypeptide as defined in
(ii)能够驱动(a)的核酸序列表达的一个或多个控制序列；和任选地(ii) one or more control sequences capable of driving the expression of the nucleic acid sequence of (a); and optionally
(iii)转录终止序列。(iii) Transcription termination sequence.
13.根据项12的构建体，其中所述控制序列之一是组成型启动子，优选GOS2启动子，最优选来自稻的GOS2启动子。13. Construct according to item 12, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
14.根据项12或13的构建体在用于制备相对于对照植物具有增加的产量，特别是增加的生物量和/或增加的种子产量的植物的方法中的用途。14. Use of the construct according to item 12 or 13 in a method for producing plants having increased yield, in particular increased biomass and/or increased seed yield relative to control plants.
15.利用根据项12或13的构建体转化的植物、植物部分或植物细胞。15. A plant, plant part or plant cell transformed with a construct according to item 12 or 13.
16.用于产生相对于对照植物具有增加的产量，特别是增加的生物量和/或增加的种子产量的转基因植物的方法，其包括：16. A method for producing a transgenic plant with increased yield, particularly increased biomass and/or increased seed production relative to control plants, comprising:
(i)向植物中引入和表达如项1或2中定义的编码CRL多肽的核酸；和(i) introducing and expressing a nucleic acid encoding a CRL polypeptide as defined in
(ii)在促进植物生长和发育的条件下培养所述植物细胞。(ii) culturing said plant cells under conditions that promote plant growth and development.
17.相对于对照植物具有增加的产量，特别是增加的生物量和/或增加的种子产量的转基因植物、或源自所述转基因植物的转基因植物细胞，其中所述增加的产量因如项1或2中定义的编码CRL样多肽的核酸的被调节的表达而产生。17. A transgenic plant with increased yield, particularly increased biomass and/or increased seed yield, or a transgenic plant cell derived from said transgenic plant, with respect to a control plant, wherein said increased yield is due to or the regulated expression of a nucleic acid encoding a CRL-like polypeptide as defined in 2.
18.根据项11、15或17的转基因植物或源自其的转基因植物细胞，其中所述植物是作物植物或单子叶植物或谷类植物例如稻、玉米、小麦、大麦、粟、黑麦、黑小麦、高粱、二粒小麦、斯佩耳特小麦、黑麦、单粒小麦、埃塞俄比亚画眉草、买罗高梁和燕麦。18. The transgenic plant or a transgenic plant cell derived therefrom according to item 11, 15 or 17, wherein said plant is a crop plant or a monocotyledonous plant or a cereal plant such as rice, maize, wheat, barley, millet, rye, rye Wheat, Sorghum, Emmer, Spelt, Rye, Einkorn, Ethiopian Teff, Milo, and Oats.
19.根据项18的植物的可收获部分，其中所述可收获部分优选是枝条生物质和/或种子。19. Harvestable parts of a plant according to item 18, wherein said harvestable parts are preferably shoot biomass and/or seeds.
20.从根据项18的植物和/或从根据项19的植物的可收获部分得到的产品。20. Products obtained from plants according to item 18 and/or from harvestable parts of plants according to item 19.
21.编码CRL多肽的核酸在相对于对照植物增加植物的产量，特别是在增加种子产量和/或枝条生物量中的用途。21. Use of a nucleic acid encoding a CRL polypeptide for increasing the yield of a plant relative to control plants, in particular for increasing seed yield and/or shoot biomass.
22.分离的核酸分子，其选自：22. An isolated nucleic acid molecule selected from the group consisting of:
(i)SEQ ID NO：195所示的核酸；(i) the nucleic acid shown in SEQ ID NO: 195;
(ii)SEQ ID NO：195所示的核酸的互补序列；(ii) the complementary sequence of the nucleic acid shown in SEQ ID NO:195;
(iii)编码SEQ ID NO：196所示的多肽的核酸，优选地作为遗传密码简并性的结果，所述分离的核酸可从SEQ ID NO：196所示的多肽序列得出，并且还优选地赋予相对于对照植物增强的产量相关性状；(iii) a nucleic acid encoding the polypeptide shown in SEQ ID NO: 196, preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from the polypeptide sequence shown in SEQ ID NO: 196, and also preferably Confers enhanced yield-related traits relative to control plants;
(iv)核酸，其按照递增的优选次序与表A7的任何核酸序列具有至少30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且还优选地赋予相对于对照植物增强的产量相关性状；(iv) a nucleic acid having at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56% , 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and preferably also confers enhanced yield-related traits relative to control plants;
(v)核酸分子，其在严格杂交条件下与(i)至(iv)的核酸分子杂交并且优选赋予相对于对照植物增强的产量相关性状；(v) nucleic acid molecule, it hybridizes under stringent hybridization condition with the nucleic acid molecule of (i) to (iv) and preferably confers the yield-related character that strengthens with respect to control plant;
(vi)编码ASPAT多肽的核酸，所述多肽按照递增的优选次序与SEQ ID NO：196所示的氨基酸序列或表A7中任何其他氨基酸序列具有至少50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且优选赋予相对于对照植物增强的产量相关性状。(vi) a nucleic acid encoding an ASPAT polypeptide having at least 50%, 51%, 52%, 53%, in ascending order of preference with the amino acid sequence shown in SEQ ID NO: 196 or any other amino acid sequence in Table A7 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and preferably confers enhanced relative to control plants yield-related traits.
23.分离的多肽，其选自：23. An isolated polypeptide selected from the group consisting of:
(i)SEQ ID NO：196所示的氨基酸序列；(i) the amino acid sequence shown in SEQ ID NO: 196;
(ii)氨基酸序列，其按照递增的优选次序与SEQ ID NO：196所示的氨基酸序列或表A7中任何其他氨基酸序列具有至少50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且优选赋予相对于对照植物增强的产量相关性状；(ii) an amino acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55% of the amino acid sequence shown in SEQ ID NO: 196 or any other amino acid sequence in Table A7 in increasing order of preference , 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72 %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and preferably confers enhanced yield-related traits relative to control plants;
(iii)上述(i)或(ii)所给出的任何氨基酸序列的衍生物。(iii) Derivatives of any of the amino acid sequences given in (i) or (ii) above.
附图说明Description of drawings
现参考以下附图描述本发明，其中：The invention will now be described with reference to the following drawings, in which:
图1显示双元载体，用于在稻GOS2启动子(pGOS2)控制之下增加alfin-样编码核酸在稻(Oryza sativa)中的表达。Figure 1 shows a binary vector for increased expression of an alfin-like encoding nucleic acid in Oryza sativa under the control of the rice GOS2 promoter (pGOS2).
图2显示双元载体，用于在稻GOS2启动子(pGOS2)控制之下增加YRP编码核酸在稻中的表达。Figure 2 shows a binary vector for increased expression of a YRP-encoding nucleic acid in Oryza sativa under the control of the rice GOS2 promoter (pGOS2).
图3显示油菜素类固醇生物合成和生长素信号转导是如何通过反馈环而连接的，根据Mouchel等(2006)Nature 443：458-461，这涉及BRX(最佳根生长所需)。Figure 3 shows how brassinosteroid biosynthesis and auxin signaling are linked through a feedback loop, which involves BRX (required for optimal root growth) according to Mouchel et al. (2006) Nature 443:458-461.
图4显示如SEQ ID NO：18所示的BRXL多肽的卡通图，其包含下列特征：(i)代表BRX结构域的保守结构域1，其包含IPR013591 DZC结构域(PFAM登录号PF08381 DZC)；(2)代表BRX结构域的保守结构域2，其包含C末端IPR013591 DZC结构域(PFAM登录号PF08381 DZC)；(3)保守结构域3和(4)保守结构域4，两者都包含保守的Cys，其间隔指示潜在的锌结合基序。Figure 4 shows a cartoon of the BRXL polypeptide as shown in SEQ ID NO: 18, which comprises the following features: (i) represents the conserved
图5显示来自表A3的BRXL多肽的ClustalW 1.81多重序列比对。下列特征加框突显：(i)代表BRX结构域的保守结构域1，其包含IPR013591DZC结构域(PFAM登录号PF08381 DZC；用X标记)；(2)代表BRX结构域的保守结构域2，其包含C末端IPR013591 DZC结构域(PFAM登录号PF08381 DZC；用X标记)；(3)保守结构域3和(4)保守结构域4，两者都包含保守的Cys(加浅色框的)，其间隔指示潜在的锌结合基序。Figure 5 shows the ClustalW 1.81 multiple sequence alignment of the BRXL polypeptides from Table A3. The following features are boxed and highlighted: (i) represents the conserved
图6显示双元载体，用于在于植物中起作用的组成型启动子控制之下增加编码BRXL多肽的核酸序列在稻植物中的表达。Figure 6 shows a binary vector for increased expression in rice plants of a nucleic acid sequence encoding a BRXL polypeptide under the control of a constitutive promoter functional in plants.
图7显示双元载体，用于在稻的GOS2启动子(pGOS2)控制之下增加silky-1样编码核酸在稻中的表达。Figure 7 shows a binary vector for increased expression of a silky-1-like encoding nucleic acid in Oryza sativa under the control of the Oryza sativa GOS2 promoter (pGOS2).
图8显示ARP6多肽的多重比对。Figure 8 shows a multiple alignment of ARP6 polypeptides.
图9显示由Kandasamy等2004描述的ARP多肽的系统发生树。指示了ARP6多肽的组。Figure 9 shows the phylogenetic tree of ARP polypeptides described by Kandasamy et al. 2004. Groups of ARP6 polypeptides are indicated.
图10显示双元载体，用于在稻GOS2启动子(pGOS2)控制之下增加ARP6编码核酸在稻中的表达。Figure 10 shows a binary vector for increased expression of an ARP6-encoding nucleic acid in Oryza sativa under the control of the rice GOS2 promoter (pGOS2).
图11显示SEQ ID NO：117，其中分别以带下划线的斜体字和粗体字标出保守的DPPIV_N和肽酶S9结构域。Figure 11 shows SEQ ID NO: 117, wherein the conserved DPPIV_N and peptidase S9 domains are indicated in underlined italics and bold, respectively.
图12显示多种POP序列的多重比对。Figure 12 shows a multiple alignment of various POP sequences.
图13显示脯氨酰肽酶的系统发生树(Tripathi&Sowdhamini，2006)。具有SEQ ID NO：117和其稻直向同源物的分支用箭头标示。Figure 13 shows a phylogenetic tree of prolyl peptidases (Tripathi & Sowdhamini, 2006). Branches with SEQ ID NO: 117 and its rice orthologues are indicated by arrows.
图14显示双元载体，用于在稻GOS2启动子(pGOS2)控制之下增加POP编码核酸在稻中的表达。Figure 14 shows a binary vector for increased expression of a POP-encoding nucleic acid in Oryza sativa under the control of the rice GOS2 promoter (pGOS2).
图15显示CRL蛋白的多重比对。Figure 15 shows a multiple alignment of CRL proteins.
图16显示CRL蛋白的系统发生树。显示了双子叶植物、单子叶植物和其他植物界CRL蛋白的聚簇。Figure 16 shows a phylogenetic tree of CRL proteins. Clustering of CRL proteins in dicots, monocots and other plant kingdoms is shown.
图17显示双元载体，用于在稻GOS2启动子(pGOS2)控制之下增加CRL编码核酸在稻中的表达。Figure 17 shows a binary vector for increased expression of a CRL-encoding nucleic acid in Oryza sativa under the control of the rice GOS2 promoter (pGOS2).
实施例Example
现参考以下实施例描述本发明，所述实施例仅意在举例说明。如下实施例并非旨在完全限定或以其他方式限制本发明的范围。The invention will now be described with reference to the following examples, which are intended to be illustrative only. The following examples are not intended to fully define or otherwise limit the scope of the invention.
DNA操作：除非另外说明，重组DNA技术根据描述于(Sambrook(2001)《分子克隆：实验室手册》，第三版，冷泉港实验室出版，冷泉港，纽约)或者Ausubel等(1994)，Current Protocols in Molecular Biology，CurrentProtocols第一卷和第二卷的标准方案进行。用于植物分子工作的标准材料和方法由R.D.D.Croy描述于Plant Molecular Biology Labfase(1993)，由BIOS Scientific Publications Ltd(UK)和Blackwell Scientific Publications(UK)出版。DNA manipulations: Unless otherwise stated, recombinant DNA techniques were used as described in (Sambrook (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, NY) or Ausubel et al. (1994), Current Protocols in Molecular Biology,
1.1.Alfin样多肽1.1. Alfin-like peptides
利用数据库序列搜索工具，例如基本局部比对工具(BLAST)(Altschul等(1990)J.Mol.Biol.215：403-410；和Altschul等(1997)Nucleic Acids Res.25：3389-3402)，在美国国家生物技术信息中心(NCBI)Entrez核苷酸数据库所保持的序列中，鉴定与SEQ ID NO：1和SEQ ID NO：3相关的序列(全长cDNA、EST或基因组序列)。该程序通过将核酸或多肽序列与序列数据库进行比较，以及通过计算匹配的统计学显著性，用于寻找序列之间的局部相似的区域。例如，在TBLASTN算法，利用SEQ ID NO：1和SEQ IDNO：3的核酸所编码的多肽，其中使用默认设置，开启过滤器以忽略低复杂度序列。分析的输出视窗为两两比较，并根据概率分值(E值)排序，其中分值反映特定比对偶然发生的概率(E值越低，命中事件的显著性越高)。除了E值之外，还对比较进行同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在一些情况下，可调整缺省参数来改变搜索的严格性。例如增加E值以显示不太严格的匹配。这样，可鉴定到短的几乎完全的匹配。Using database sequence search tools such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Sequences (full-length cDNA, EST or genomic sequences) related to SEQ ID NO: 1 and SEQ ID NO: 3 were identified in sequences maintained by the National Center for Biotechnology Information (NCBI) Entrez nucleotide database. This program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of the matches. For example, in the TBLASTN algorithm, the polypeptides encoded by the nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 3 are used, wherein default settings are used, and the filter is turned on to ignore low-complexity sequences. The output window of the analysis is a pairwise comparison and is ordered according to the probability score (E-value), where the score reflects the probability that a particular alignment occurred by chance (the lower the E-value, the more significant the hit event). In addition to E-values, comparisons are scored for percent identity. The percent identity refers to the number of identical nucleotides (or amino acids) over a specific length between two compared nucleic acid (or polypeptide) sequences. In some cases, the default parameters can be adjusted to change the stringency of the search. For example increasing the E value to show less strict matches. In this way, short almost perfect matches can be identified.
表A1提供了一列alfin样核酸序列。Table A1 provides a list of alfin-like nucleic acid sequences.
表A1：alfin样多肽的实例：Table A1: Examples of alfin-like polypeptides:
在一些情况下，相关序列已经由研究机构如基因组研究机构(Institutefor Genomic Research，TIGR)尝试性地进行了装配并向公众公开。可以通过关键词搜索，或是采用BLAST算法，运用目的核酸序列或多肽序列，利用真核基因直向同源物(Eukaryotic Gene Orthologs，EGO)数据库来鉴定这样的相关序列。在其他情况下，针对特定的生物，例如由联合基因组研究所(Joint Genome Institute)，创建了特定的核酸序列数据库。In some cases, related sequences have been tentatively assembled and released to the public by research institutions such as the Institute for Genomic Research (TIGR). Such related sequences can be identified by keyword search, or using the BLAST algorithm, using the target nucleic acid sequence or polypeptide sequence, and using the Eukaryotic Gene Orthologs (EGO) database. In other cases, specific nucleic acid sequence databases have been created for specific organisms, such as by the Joint Genome Institute.
1.2.YRP多肽1.2. YRP polypeptide
利用数据库序列搜索工具，例如基本局部比对工具(BLAST)(Altschul等(1990)J.Mol.Biol.215：403-410；和Altschul等(1997)Nucleic Acids Res.25：3389-3402)，在美国国家生物技术信息中心(NCBI)Entrez核苷酸数据库所保持的序列中，鉴定了与SEQ ID NO：10和SEQ ID NO：12相关的序列(全长cDNA、EST或基因组序列)。该程序通过将核酸或多肽序列与序列数据库进行比较，以及通过计算匹配的统计学显著性，用于寻找序列之间的局部相似的区域。例如，已经对由SEQ ID NO：10和SEQ ID NO：12的核酸所编码的多肽运用TBLASTN算法，其中使用默认设置，开启过滤器以忽略低复杂度序列。分析的输出视窗为两两比较，并根据概率分值(E值)排序，其中分值反映特定比对偶然发生的概率(E值越低，命中事件的显著性越高)。除了E值之外，还对比较进行了同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在一些情况下，可调整缺省参数来改变搜索的严格性。例如增加E值以显示不太严格的匹配。这样，可鉴定到短的几乎完全的匹配。Using database sequence search tools such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Sequences (full-length cDNA, EST or genomic sequences) related to SEQ ID NO: 10 and SEQ ID NO: 12 were identified in sequences maintained by the National Center for Biotechnology Information (NCBI) Entrez nucleotide database. This program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of the matches. For example, the TBLASTN algorithm has been applied to polypeptides encoded by the nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 12, with default settings and a filter turned on to ignore low complexity sequences. The output window of the analysis is a pairwise comparison and is ordered according to the probability score (E-value), where the score reflects the probability that a particular alignment occurred by chance (the lower the E-value, the more significant the hit event). In addition to E-values, comparisons were scored as percent identity. The percent identity refers to the number of identical nucleotides (or amino acids) over a specific length between two compared nucleic acid (or polypeptide) sequences. In some cases, the default parameters can be adjusted to change the stringency of the search. For example increasing the E value to show less strict matches. In this way, short almost perfect matches can be identified.
表A2提供了YRP核酸序列的列表。Table A2 provides a list of YRP nucleic acid sequences.
表A2：YRP多肽的实例：Table A2: Examples of YRP polypeptides:
在一些情况下，相关序列已经由研究机构如基因组研究机构(TIGR；始于TA)尝试性地进行了装配并向公众公开。可以通过关键词搜索，或是采用BLAST算法，运用目的核酸序列或多肽序列，利用真核基因直向同源物(EGO)数据库来鉴定这样的相关序列。在其他情况下，已经针对特定的生物创建了特定的核酸序列数据库，例如由联合基因组研究所创建。In some cases, related sequences have been tentatively assembled and released to the public by research institutions such as the Institute for Genome Research (TIGR; from TA). Such related sequences can be identified by keyword searching, or using the BLAST algorithm, using the nucleic acid sequence or polypeptide sequence of interest, using the eukaryotic gene ortholog (EGO) database. In other cases, specific nucleic acid sequence databases have been created for specific organisms, such as by the Joint Genome Institute.
1.3.Brevis Radix样(BRXL)多肽1.3. Brevis Radix-like (BRXL) polypeptide
利用数据库序列搜索工具，例如基本局部比对工具(BLAST)(Altschul等(1990)J.Mol.Biol.215：403-410；和Altschul等(1997)Nucleic Acids Res.25：3389-3402)，在美国国家生物技术信息中心(NCBI)Entrez核苷酸数据库所保持的序列中，鉴定了与用于本发明的方法中的核酸序列相关的序列(全长cDNA、EST或基因组序列)。该程序通过将核酸或多肽序列与序列数据库进行比较，以及通过计算匹配的统计学显著性，用于寻找序列之间的局部相似的区域。例如，已经对由本发明的核酸序列所编码的多肽运用TBLASTN算法，其中使用默认设置，开启过滤器以忽略低复杂度序列。分析的输出视窗为两两比较，并根据概率分值(E值)排序，其中分值反映特定比对偶然发生的概率(E值越低，命中事件的显著性越高)。除了E值之外，还对比较进行了同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在一些情况下，可调整缺省参数来改变搜索的严格性。例如增加E值以显示不太严格的匹配。这样，可鉴定到短的几乎完全的匹配。Using database sequence search tools such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Sequences (full-length cDNA, EST or genomic sequences) related to the nucleic acid sequences used in the methods of the invention are identified in the sequences maintained by the National Center for Biotechnology Information (NCBI) Entrez nucleotide database. This program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of the matches. For example, the TBLASTN algorithm has been applied to polypeptides encoded by nucleic acid sequences of the invention with default settings and a filter turned on to ignore sequences of low complexity. The output window of the analysis is a pairwise comparison and is ordered according to the probability score (E-value), where the score reflects the probability that a particular alignment occurred by chance (the lower the E-value, the more significant the hit event). In addition to E-values, comparisons were scored as percent identity. The percent identity refers to the number of identical nucleotides (or amino acids) over a specific length between two compared nucleic acid (or polypeptide) sequences. In some cases, the default parameters can be adjusted to change the stringency of the search. For example increasing the E value to show less strict matches. In this way, short almost perfect matches can be identified.
表A3提供了与本发明方法中所用的核酸序列相关的核酸序列的列表。Table A3 provides a list of nucleic acid sequences related to the nucleic acid sequences used in the methods of the invention.
表A3：BRXL多肽序列和编码核酸序列的实例Table A3: Examples of BRXL polypeptide sequences and encoding nucleic acid sequences
在一些情况下，相关序列已经由研究机构如基因组研究机构(TIGR；始于TA)或Genoscope(始于GS)尝试性地进行了装配并向公众公开。可以通过关键词搜索，或是采用BLAST算法，运用目的核酸序列或多肽序列，利用真核基因直向同源物(EGO)数据库来鉴定这样的相关序列。在其他情况下，已经针对特定的生物创建了特定的核酸序列数据库，例如由联合基因组研究所创建。此外，访问专利数据库(proprietary databases)也允许鉴定新型核酸和多肽序列。In some cases, related sequences have been tentatively assembled and released to the public by research institutions such as the Institute for Genome Research (TIGR; from TA) or Genoscope (from GS). Such related sequences can be identified by keyword searching, or using the BLAST algorithm, using the nucleic acid sequence or polypeptide sequence of interest, using the eukaryotic gene ortholog (EGO) database. In other cases, specific nucleic acid sequence databases have been created for specific organisms, such as by the Joint Genome Institute. In addition, access to proprietary databases also allows the identification of novel nucleic acid and polypeptide sequences.
1.4.silky-1样多肽1.4. silky-1-like polypeptide
利用数据库序列搜索工具，例如基本局部比对工具(BLAST)(Altschul等(1990)J.Mol.Biol.215：403-410；和Altschul等(1997)Nucleic Acids Res.25：3389-3402)，在美国国家生物技术信息中心(NCBI)Entrez核苷酸数据库所保持的序列中，鉴定了与SEQ ID NO：90、SEQ ID NO：92或SEQ ID NO：94相关的序列(全长cDNA、EST或基因组序列)。该程序通过将核酸或多肽序列与序列数据库进行比较，以及通过计算匹配的统计学显著性，用于寻找序列之间的局部相似的区域。例如，已经对由SEQ ID NO：90、SEQ IDNO：92或SEQ ID NO：94的核酸所编码的多肽运用TBLASTN算法，其中使用默认设置，开启过滤器以忽略低复杂度序列。分析的输出视窗为两两比较，并根据概率分值(E值)排序，其中分值反映特定比对偶然发生的概率(E值越低，命中事件的显著性越高)。除了E值之外，还对比较进行了同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在一些情况下，可调整缺省参数来改变搜索的严格性。例如增加E值以显示不太严格的匹配。这样，可鉴定到短的几乎完全的匹配。Using database sequence search tools such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Among the sequences maintained by the National Center for Biotechnology Information (NCBI) Entrez nucleotide database, sequences related to SEQ ID NO: 90, SEQ ID NO: 92, or SEQ ID NO: 94 were identified (full-length cDNA, EST or genome sequence). This program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of the matches. For example, the TBLASTN algorithm has been applied to polypeptides encoded by the nucleic acids of SEQ ID NO: 90, SEQ ID NO: 92, or SEQ ID NO: 94, with default settings and a filter turned on to ignore low complexity sequences. The output window of the analysis is a pairwise comparison and is ordered according to the probability score (E-value), where the score reflects the probability that a particular alignment occurred by chance (the lower the E-value, the more significant the hit event). In addition to E-values, comparisons were scored as percent identity. The percent identity refers to the number of identical nucleotides (or amino acids) over a specific length between two compared nucleic acid (or polypeptide) sequences. In some cases, the default parameters can be adjusted to change the stringency of the search. For example increasing the E value to show less strict matches. In this way, short almost perfect matches can be identified.
表A4提供了silky-1样核酸序列的列表。Table A4 provides a list of silky-1-like nucleic acid sequences.
表A4：silky-1样多肽的实例：Table A4: Examples of silky-1-like polypeptides:
在一些情况下，相关序列已经由研究机构如基因组研究机构(TIGR；始于TA)尝试性地进行了装配并向公众公开。可以通过关键词搜索，或是采用BLAST算法，运用目的核酸序列或多肽序列，利用真核基因直向同源物(EGO)数据库来鉴定这样的相关序列。在其他情况下，已经针对特定的生物创建了特定的核酸序列数据库，例如由联合基因组研究创建。In some cases, related sequences have been tentatively assembled and released to the public by research institutions such as the Institute for Genome Research (TIGR; from TA). Such related sequences can be identified by keyword searching, or using the BLAST algorithm, using the nucleic acid sequence or polypeptide sequence of interest, using the eukaryotic gene ortholog (EGO) database. In other cases, specific nucleic acid sequence databases have been created for specific organisms, for example by Consortium Genome Research.
1.5.ARP6多肽1.5. ARP6 polypeptide
利用数据库序列搜索工具，例如基本局部比对工具(BLAST)(Altschul等(1990)J.Mol.Biol.215：403-410；和Altschul等(1997)Nucleic Acids Res.25：3389-3402)，在美国国家生物技术信息中心(NCBI)Entrez核苷酸数据库所保持的序列中，鉴定了与用于本发明的方法的核酸序列相关的序列(全长cDNA、EST或基因组序列)。该程序通过将核酸或多肽序列与序列数据库进行比较，以及通过计算匹配的统计学显著性，用于寻找序列之间的局部相似的区域。例如，已经对由用于本发明的核酸所编码的多肽运用TBLASTN算法，其中使用默认设置，开启过滤器以忽略低复杂度序列。分析的输出视窗为两两比较，并根据概率分值(E值)排序，其中分值反映特定比对偶然发生的概率(E值越低，命中事件的显著性越高)。除了E值之外，还对比较进行了同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在一些情况下，可调整缺省参数来改变搜索的严格性。例如增加E值以显示不太严格的匹配。这样，可鉴定到短的几乎完全的匹配。Using database sequence search tools such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Sequences (full-length cDNA, EST or genomic sequences) related to the nucleic acid sequences used in the methods of the invention were identified in the sequences maintained by the National Center for Biotechnology Information (NCBI) Entrez nucleotide database. This program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of the matches. For example, the TBLASTN algorithm has been applied to polypeptides encoded by nucleic acids used in the present invention with default settings and a filter turned on to ignore low complexity sequences. The output window of the analysis is a pairwise comparison and is ordered according to the probability score (E-value), where the score reflects the probability that a particular alignment occurred by chance (the lower the E-value, the more significant the hit event). In addition to E-values, comparisons were scored as percent identity. The percent identity refers to the number of identical nucleotides (or amino acids) over a specific length between two compared nucleic acid (or polypeptide) sequences. In some cases, the default parameters can be adjusted to change the stringency of the search. For example increasing the E value to show less strict matches. In this way, short almost perfect matches can be identified.
表A5提供了与本发明方法中所用的核酸序列相关的核酸序列的列表。Table A5 provides a list of nucleic acid sequences related to the nucleic acid sequences used in the methods of the invention.
表A5：ARP6核酸和其编码的多肽的实例：Table A5: Examples of ARP6 nucleic acids and their encoded polypeptides:
在一些情况下，相关序列已经由研究机构如基因组研究机构(TIGR；始于TA)尝试性地进行了装配并向公众公开。可以通过关键词搜索，或是采用BLAST算法，运用目的核酸序列或多肽序列，利用真核基因直向同源物(EGO)数据库来鉴定这样的相关序列。在其他情况下，已经针对特定的生物创建了特定的核酸序列数据库，例如由联合基因组研究所创建。此外，访问专利数据库已允许鉴定新型核酸和多肽序列。In some cases, related sequences have been tentatively assembled and released to the public by research institutions such as the Institute for Genome Research (TIGR; from TA). Such related sequences can be identified by keyword searching, or using the BLAST algorithm, using the nucleic acid sequence or polypeptide sequence of interest, using the eukaryotic gene ortholog (EGO) database. In other cases, specific nucleic acid sequence databases have been created for specific organisms, such as by the Joint Genome Institute. In addition, access to patent databases has allowed the identification of novel nucleic acid and polypeptide sequences.
1.6.POP多肽1.6. POP polypeptide
利用数据库序列搜索工具，例如基本局部比对工具(BLAST)(Altschul等(1990)J.Mol.Biol.215：403-410；和Altschul等(1997)Nucleic Acids Res.25：3389-3402)，在美国国家生物技术信息中心(NCBI)Entrez核苷酸数据库所保持的序列中，鉴定了与用于本发明的方法的核酸序列相关的序列(全长cDNA、EST或基因组序列)。该程序通过将核酸或多肽序列与序列数据库进行比较，以及通过计算匹配的统计学显著性，用于寻找序列之间的局部相似的区域。例如，已经对由用于本发明的核酸所编码的多肽运用TBLASTN算法，其中使用默认设置，开启过滤器以忽略低复杂度序列。分析的输出视窗为两两比较，并根据概率分值(E值)排序，其中分值反映特定比对偶然发生的概率(E值越低，命中事件的显著性越高)。除了E值之外，还对比较进行了同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在一些情况下，可调整缺省参数来改变搜索的严格性。例如增加E值以显示不太严格的匹配。这样，可鉴定到短的几乎完全的匹配。Using database sequence search tools such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Sequences (full-length cDNA, EST or genomic sequences) related to the nucleic acid sequences used in the methods of the invention were identified in the sequences maintained by the National Center for Biotechnology Information (NCBI) Entrez nucleotide database. This program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of the matches. For example, the TBLASTN algorithm has been applied to polypeptides encoded by nucleic acids used in the present invention with default settings and a filter turned on to ignore low complexity sequences. The output window of the analysis is a pairwise comparison and is ordered according to the probability score (E-value), where the score reflects the probability that a particular alignment occurred by chance (the lower the E-value, the more significant the hit event). In addition to E-values, comparisons were scored as percent identity. The percent identity refers to the number of identical nucleotides (or amino acids) over a specific length between two compared nucleic acid (or polypeptide) sequences. In some cases, the default parameters can be adjusted to change the stringency of the search. For example increasing the E value to show less strict matches. In this way, short almost perfect matches can be identified.
表A6提供了与本发明方法中所用的核酸序列相关的核酸序列的列表。Table A6 provides a list of nucleic acid sequences related to the nucleic acid sequences used in the methods of the invention.
表A6：POP多肽的实例：Table A6: Examples of POP polypeptides:
在一些情况下，相关序列已经由研究机构如基因组研究机构(TIGR；始于TA)尝试性地进行了装配并向公众公开。可以通过关键词搜索，或是采用BLAST算法，运用目的核酸序列或多肽序列，利用真核基因直向同源物(EGO)数据库来鉴定这样的相关序列。在其他情况下，已经针对特定的生物创建了特定的核酸序列数据库，例如由联合基因组研究所创建。此外，访问专利数据库已允许鉴定新型核酸和多肽序列。In some cases, related sequences have been tentatively assembled and released to the public by research institutions such as the Institute for Genome Research (TIGR; from TA). Such related sequences can be identified by keyword searching, or using the BLAST algorithm, using the nucleic acid sequence or polypeptide sequence of interest, using the eukaryotic gene ortholog (EGO) database. In other cases, specific nucleic acid sequence databases have been created for specific organisms, such as by the Joint Genome Institute. In addition, access to patent databases has allowed the identification of novel nucleic acid and polypeptide sequences.
1.7.Crumpled Leaf(CRL)多肽1.7. Crumpled Leaf (CRL) peptide
利用数据库序列搜索工具，例如基本局部比对工具(BLAST)(Altschul等(1990)J.Mol.Biol.215：403-410；和Altschul等(1997)Nucleic Acids Res.25：3389-3402)，在美国国家生物技术信息中心(NCBI)Entrez核苷酸数据库所保持的序列中，鉴定了与用于本发明方法的核酸序列相关的序列(全长cDNA、EST或基因组序列)。该程序通过将核酸或多肽序列与序列数据库进行比较，以及通过计算匹配的统计学显著性，用于寻找序列之间的局部相似的区域。例如，已经对由用于本发明的核酸所编码的多肽运用TBLASTN算法，其中使用默认设置，开启过滤器以忽略低复杂度序列。分析的输出视窗为两两比较，并根据概率分值(E值)排序，其中分值反映特定比对偶然发生的概率(E值越低，命中事件的显著性越高)。除了E值之外，还对比较进行了同一性百分比记分。同一性百分比是指两比较核酸(或多肽)序列之间在特定长度上的相同核苷酸(或氨基酸)数。在一些情况下，可调整缺省参数来改变搜索的严格性。例如增加E值以显示不太严格的匹配。这样，可鉴定到短的几乎完全的匹配。Using database sequence search tools such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Sequences (full-length cDNA, EST or genomic sequences) related to the nucleic acid sequences used in the methods of the invention were identified in the sequences maintained by the National Center for Biotechnology Information (NCBI) Entrez nucleotide database. This program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of the matches. For example, the TBLASTN algorithm has been applied to polypeptides encoded by nucleic acids used in the present invention with default settings and a filter turned on to ignore low complexity sequences. The output window of the analysis is a pairwise comparison and is ordered according to the probability score (E-value), where the score reflects the probability that a particular alignment occurred by chance (the lower the E-value, the more significant the hit event). In addition to E-values, comparisons were scored as percent identity. The percent identity refers to the number of identical nucleotides (or amino acids) over a specific length between two compared nucleic acid (or polypeptide) sequences. In some cases, the default parameters can be adjusted to change the stringency of the search. For example increasing the E value to show less strict matches. In this way, short almost perfect matches can be identified.
表A7提供了与本发明方法中所用的核酸序列相关的核酸序列的列表。Table A7 provides a list of nucleic acid sequences related to the nucleic acid sequences used in the methods of the invention.
表A7：CRL多肽的实例：Table A7: Examples of CRL polypeptides:
在一些情况下，相关序列已经由研究机构如基因组研究机构(TIGR；始于TA)尝试性地进行了装配并向公众公开。可以通过关键词搜索，或是采用BLAST算法，运用目的核酸序列或多肽序列，利用真核基因直向同源物(EGO)数据库来鉴定这样的相关序列。在其他情况下，已经针对特定的生物创建了特定的核酸序列数据库，例如由联合基因组研究所(JointGenome Institute)创建。访问专利数据库已允许鉴定新型核酸和多肽序列。In some cases, related sequences have been tentatively assembled and released to the public by research institutions such as the Institute for Genome Research (TIGR; from TA). Such related sequences can be identified by keyword searching, or using the BLAST algorithm, using the nucleic acid sequence or polypeptide sequence of interest, using the eukaryotic gene ortholog (EGO) database. In other cases, specific nucleic acid sequence databases have been created for specific organisms, such as by the Joint Genome Institute. Access to patent databases has allowed the identification of novel nucleic acid and polypeptide sequences.
实施例2：与用于本发明的方法的多肽序列相关的序列的比对Example 2: Alignment of sequences related to polypeptide sequences used in the methods of the invention
2.1.Alfin样多肽2.1. Alfin-like peptides
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet(或Blosum 62(如果比对多肽)，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet (or Blosum 62 (if comparing peptides), gap opening penalty: 10, gap extension penalty: 0.2) for peptide sequence alignment. Minor manual edits are made to further optimize the alignment.
使用Vector NTI(Invitrogen)的AlignX程序中提供的邻接聚类算法(neighbour-joining clustering algorithm)，构建alfin样多肽的系统发生树。A phylogenetic tree of alfin-like peptides was constructed using the neighbor-joining clustering algorithm provided in the AlignX program of Vector NTI (Invitrogen).
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet, gap opening penalty: 10, gap extension penalty: 0.2) to compare polypeptide sequences. Minor hand edits were made to further refine the alignment.
2.2.YRP多肽2.2. YRP polypeptide
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet(或Blosum 62(如果比对多肽)，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet (or Blosum 62 (if comparing peptides), gap opening penalty: 10, gap extension penalty: 0.2) for peptide sequence alignment. Minor manual edits are made to further optimize the alignment.
使用Vector NTI(Invitrogen)的AlignX程序中提供的邻接聚类算法，构建了YRP多肽的系统发生树。A phylogenetic tree of YRP polypeptides was constructed using the neighbor-joining clustering algorithm provided in the AlignX program of Vector NTI (Invitrogen).
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet, gap opening penalty: 10, gap extension penalty: 0.2) to compare polypeptide sequences. Minor hand edits were made to further refine the alignment.
2.3.Brevis Radix样(BRXL)多肽2.3. Brevis Radix-like (BRXL) polypeptide
表A3中的所有BRXL多肽序列的多重序列比对利用ClustalW 1.81算法进行。比对结果示于本申请的图5中。下列特征加框突显：(i)表示BRX结构域的保守结构域1，其包括IPR013591 DZC结构域(PFAM登录号PF08381 DZC；用X标记)；(2)表示BRX结构域的保守结构域2，其包括C末端IPR013591 DZC结构域(PFAM登录号PF08381 DZC；用X标记)；(3)保守结构域3和(4)保守结构域4，两者都包含保守Cys(加淡框的)，其间隔指示潜在的锌结合基序。Multiple sequence alignments of all BRXL polypeptide sequences in Table A3 were performed using the ClustalW 1.81 algorithm. The results of the alignment are shown in Figure 5 of the present application. The following features are boxed and highlighted: (i) represents the conserved
2.4.silky-1样多肽2.4. silky-1-like polypeptide
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet(或Blosum62(如果比对多肽)，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet (or Blosum62 (if comparing polypeptides), gap opening penalty: 10, gap extension penalty: 0.2) for polypeptide sequence alignment. Minor manual edits were made to further optimize the alignment.
使用Vector NTI(Invitrogen)的AlignX程序中提供的邻接聚类算法，构建silky-1样多肽的系统发生树。A phylogenetic tree of silky-1-like polypeptides was constructed using the neighbor-joining clustering algorithm provided in the AlignX program of Vector NTI (Invitrogen).
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet, gap opening penalty: 10, gap extension penalty: 0.2) to compare polypeptide sequences. Minor hand edits were made to further refine the alignment.
2.5.ARP6多肽2.5. ARP6 polypeptide
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet(或Blosum 62(如果比对多肽)，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。在图8中比对ARP6多肽。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet (or Blosum 62 (if comparing polypeptides), gap opening penalty: 10, gap extension penalty: 0.2) for polypeptide sequence alignment. Minor manual edits were made to further optimize the alignment. Compare in Figure 8 to the ARP6 polypeptide.
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet, gap opening penalty: 10, gap extension penalty: 0.2) to compare polypeptide sequences. Minor hand edits were made to further refine the alignment.
2.6.POP多肽2.6. POP polypeptide
利用渐近比对的ClustalW 2.0算法(Thompson等(1997)Nucleic AcidsRes 25：4876-4882；Chenna等(2003).Nucleic Acids Res 31：3497-3500)，使用标准设置(slow比对，相似性矩阵：Gonnet，空位开放罚分10，空位延伸罚分：0.2)来进行多肽序列的比对。进行微小的手工编辑以进一步优化比对。在图12中比对POP多肽。ClustalW 2.0 algorithm using asymptotic alignment (Thompson et al. (1997) Nucleic Acids Res 25: 4876-4882; Chenna et al. (2003). Nucleic Acids Res 31: 3497-3500), using standard settings (slow alignment, similarity matrix : Gonnet, gap opening penalty: 10, gap extension penalty: 0.2) to compare polypeptide sequences. Minor hand edits were made to further refine the alignment. The POP polypeptides are aligned in FIG. 12 .
如下构建POP多肽的系统发生树(图3，Tripathi&Sowdhamini，2006)：A phylogenetic tree of POP polypeptides was constructed as follows (Figure 3, Tripathi & Sowdhamini, 2006):
使用CLUSTALW程序，构建了丝氨酸蛋白酶结构域的多重序列比对。为了比较等同区域，利用HMMALIGN(描绘针对PfamA数据库实施的匹配方法的一个序列)，检索结构域区域。比对不包括缺乏该蛋白酶样结构域的显著部分的蛋白质。使用Blosum 30矩阵、空位开放罚分10和空位延伸罚分：0.05。利用PHYLIP(Phylogeny Inference Package)3.65，从多重序列比对推出了总体系统发生树。使用SEQBOOT进行了100次自展(Bootstrapping)，以针对每一个内部分支获得支持值(support value)。利用PROTDIST确定成对距离。利用NEIGHBOR，使用标准参照，计算邻接系统发生树(Neighbourjoining phylogenetic trees)。利用程序CONSENSE获得全部自展序列(bootstrapped sequences)的多数原则一致性树(majority-rule consensus tree)。使用TreeView构建所计算的树的代表。具有大于50％的自展值的聚簇被定义为确认的亚组，具有较低值的序列根据它们在比对中的序列相似性(通过目检判断)而加入这些亚组。Multiple sequence alignments of serine protease domains were constructed using the CLUSTALW program. To compare equivalent regions, domain regions were searched using HMMALIGN (a sequence depicting a matching method implemented against the PfamA database). Proteins lacking a significant portion of this protease-like domain were excluded from the alignment. Using Blosum 30 matrix,
2.7.Crumpled Leaf(CRL)多肽2.7. Crumpled Leaf (CRL) peptide
使用MUSCLE 3.7算法(通过Log-Expectation的多重序列比对)(Edgard 2004of Nucleic Acids Research，2004，第32卷，No.51792-1797)，利用标准设置进行多肽序列的比对(图15)。Alignment of polypeptide sequences was performed with standard settings using the MUSCLE 3.7 algorithm (Multiple Sequence Alignment by Log-Expectation) (Edgard 2004 of Nucleic Acids Research, 2004, Vol. 32, No. 51792-1797) with standard settings ( FIG. 15 ).
使用QuickTree(Howe等(2002)，Bioinformatics 18(11)：1546-7)计算邻接树。显示了100次自展重复后主分支的支持率。使用Dendroscope(Huson等.(2007)，BMC Bioinformatics 8(1)：460)画出环形系统发生图。参见图16。CRL蛋白似乎在大多数生物中是独特的。Neighbor-joining trees were calculated using QuickTree (Howe et al. (2002), Bioinformatics 18(11): 1546-7). The support of the main branch after 100 bootstrap repetitions is shown. Circular phylogenetic diagrams were drawn using Dendroscope (Huson et al. (2007), BMC Bioinformatics 8(1):460). See Figure 16. CRL proteins appear to be unique among most organisms.
实施例3：计算可以用于实施本发明方法的多肽序列之间的全局同一性百分比Example 3: Calculating the percent global identity between polypeptide sequences that can be used to implement the methods of the invention
3.1.Alfin样多肽3.1. Alfin-like peptides
全长多肽序列之间的全局相似性和同一性百分比，利用本领域可用方法之一即MatGAT(矩阵全局比对工具)软件(BMC Bioinformatics.20034：29.MatGAT：an application that generates similarity/identity matricesusing protein or DNA sequences.Campanella JJ，Bitincka L，Smalley J；软件由Ledion Bitincka托管)来确定。MatGAT软件无需对数据进行预比对，即可产生DNA或蛋白质序列的相似性/同一性矩阵。该程序利用Myers和Miller全局比对算法(空位开放罚分为12，而空位延伸罚分为2)进行一系列的两两比对，利用例如Blosum 62(对于多肽而言)计算相似性和同一性，然后将结果排列成距离矩阵。序列相似性示于对角线下半部，而序列同一性示于对角线上半部。For the global similarity and identity percentage between full-length polypeptide sequences, one of the methods available in the art is MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics.20034: 29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella JJ, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software can generate similarity/identity matrices of DNA or protein sequences without pre-aligning the data. The program performs a series of pairwise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12 and a gap extension penalty of 2) and calculates similarity and identity using, for example, Blosum 62 (for polypeptides). , and then arrange the results into a distance matrix. Sequence similarity is shown on the lower diagonal, while sequence identity is shown on the upper diagonal.
比较所用的参数为：The parameters used for comparison are:
记分矩阵：Blosum 62Scoring Matrix: Blosum 62
首个空位：12First available slot: 12
延伸空位：2Extended Slots: 2
也可产生用于特定结构域的局部比对的MATGAT表，或关于特定结构域之间的同一性/相似性百分比的数据。MATGAT tables for local alignments of specific domains, or data regarding percent identity/similarity between specific domains can also be generated.
3.2.YRP多肽3.2. YRP polypeptide
全长多肽序列之间的全局相似性和同一性百分比，利用本领域可用方法之一即MatGAT(矩阵全局比对工具)软件(BMC Bioinformatics.20034：29.MatGAT：an application that generates similarity/identity matricesusing protein or DNA sequences.Campanella JJ，Bitincka L，Smalley J；软件由Ledion Bitincka托管)来确定。MatGAT软件无需对数据进行预比对，即可产生DNA或蛋白质序列的相似性/同一性矩阵。该程序利用Myers和Miller全局比对算法(空位开放罚分为12，而空位延伸罚分为2)进行一系列的两两比对，利用例如Blosum 62(对于多肽而言)计算相似性和同一性，然后将结果排列成距离矩阵。序列相似性示于对角线下半部，而序列同一性示于对角线上半部。For the global similarity and identity percentage between full-length polypeptide sequences, one of the methods available in the art is MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics.20034: 29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella JJ, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software can generate similarity/identity matrices of DNA or protein sequences without pre-aligning the data. The program performs a series of pairwise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12 and a gap extension penalty of 2) and calculates similarity and identity using, for example, Blosum 62 (for polypeptides). , and then arrange the results into a distance matrix. Sequence similarity is shown on the lower diagonal, while sequence identity is shown on the upper diagonal.
比较所用的参数为：The parameters used for comparison are:
记分矩阵：Blosum 62Scoring Matrix: Blosum 62
首个空位：12First available slot: 12
延伸空位：2Extended Slots: 2
也可产生用于特定结构域的局部比对的MATGAT表，或关于特定结构域之间的同一性/相似性百分比的数据。MATGAT tables for local alignments of specific domains, or data regarding percent identity/similarity between specific domains can also be generated.
3.3.Brevis Radix样(BRXL)多肽3.3. Brevis Radix-like (BRXL) polypeptide
全长多肽序列之间的全局相似性和同一性百分比，利用本领域可用方法之一即MatGAT(矩阵全局比对工具)软件(BMC Bioinformatics.20034：29.MatGAT：an application that generates similarity/identity matricesusing protein or DNA sequences.Campanella JJ，Bitincka L，Smalley J；软件由Ledion Bitincka托管)来确定。MatGAT软件无需对数据进行预比对，即可产生DNA或蛋白质序列的相似性/同一性矩阵。该程序利用Myers和Miller全局比对算法(空位开放罚分为12，而空位延伸罚分为2)进行一系列的两两比对，利用例如Blosum 62(对于多肽而言)计算相似性和同一性，然后将结果排列成距离矩阵。序列相似性示于对角线下半部，而序列同一性示于对角线上半部。For the global similarity and identity percentage between full-length polypeptide sequences, one of the methods available in the art is MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics.20034: 29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella JJ, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software can generate similarity/identity matrices of DNA or protein sequences without pre-aligning the data. The program performs a series of pairwise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12 and a gap extension penalty of 2) and calculates similarity and identity using, for example, Blosum 62 (for polypeptides). , and then arrange the results into a distance matrix. Sequence similarity is shown on the lower diagonal, while sequence identity is shown on the upper diagonal.
比较所用的参数为：The parameters used for comparison are:
记分矩阵：Blosum 62Scoring Matrix: Blosum 62
首个空位：12First available slot: 12
延伸空位：2Extended Slots: 2
多肽序列全长范围(将部分多肽序列排除在外)的全局相似性和同一性的软件分析结果示于表B1。The software analysis results of the global similarity and identity over the entire length of the polypeptide sequences (excluding partial polypeptide sequences) are shown in Table B1.
与SEQ ID NO：18相比较，可以用于实施本发明方法的全长多肽序列之间的同一性百分比可以低至43％氨基酸同一性。Compared with SEQ ID NO: 18, the percent identity between the full-length polypeptide sequences that can be used to practice the methods of the present invention can be as low as 43% amino acid identity.
如果比较多肽的最保守的区域，则氨基酸同一性百分比可显著增加。例如，当比较如SEQ ID NO：83表示的保守结构域1或如SEQ ID NO：84表示的保守结构域2的氨基酸序列与表A3多肽的各自相应结构域时，氨基酸同一性百分比显著增加(按照优选次序至少50％，55％，60％，65％，70％，75％，80％，85％，90％，95％，98％，99％或更高的氨基酸序列同一性)。The percent amino acid identity can increase significantly if the most conserved regions of polypeptides are compared. For example, when comparing the amino acid sequence of the conserved
3.4.silky-1样多肽3.4. silky-1-like polypeptide
全长多肽序列之间的全局相似性和同一性百分比，利用本领域可用方法之一即MatGAT(矩阵全局比对工具)软件(BMC Bioinformatics.2003 4：29.MatGAT：an application that generates similarity/identitymatrices using protein or DNA sequences.Campanella JJ，Bitincka L，Smalley J；软件由Ledion Bitincka托管)来确定。MatGAT软件无需对数据进行预比对，即可产生DNA或蛋白质序列的相似性/同一性矩阵。该程序利用Myers和Miller全局比对算法(空位开放罚分为12，而空位延伸罚分为2)进行一系列的两两比对，利用例如Blosum 62(对于多肽而言)计算相似性和同一性，然后将结果排列成距离矩阵。序列相似性示于对角线下半部，而序列同一性示于对角线上半部。For the global similarity and identity percentage between full-length polypeptide sequences, use one of the methods available in this field, namely MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics.2003 4: 29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella JJ, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software can generate similarity/identity matrices of DNA or protein sequences without pre-aligning the data. The program performs a series of pairwise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12 and a gap extension penalty of 2) and calculates similarity and identity using, for example, Blosum 62 (for polypeptides). , and then arrange the results into a distance matrix. Sequence similarity is shown on the lower diagonal, while sequence identity is shown on the upper diagonal.
比较所用的参数为：The parameters used for comparison are:
记分矩阵：Blosum 62Scoring Matrix: Blosum 62
首个空位：12First available slot: 12
延伸空位：2Extended Slots: 2
也可产生用于特定结构域的局部比对的MATGAT表，或关于特定结构域之间的同一性/相似性百分比的数据。MATGAT tables for local alignments of specific domains, or data regarding percent identity/similarity between specific domains can also be generated.
3.5.ARP63.5. ARP6
可以用于实施本发明方法的全长多肽序列之间的全局相似性和同一性百分比，利用本领域可用方法之一即MatGAT(矩阵全局比对工具)软件(BMC Bioinformatics.20034：29.MatGAT：an application thatgenerates similarity/identity matrices using protein or DNA sequences.Campanella JJ，Bitincka L，Smalley J；软件由Ledion Bitincka托管)来确定。MatGAT软件无需对数据进行预比对，即可产生DNA或蛋白质序列的相似性/同一性矩阵。该程序利用Myers和Miller全局比对算法(空位开放罚分为12，而空位延伸罚分为2)进行一系列的两两比对，利用例如Blosum 62(对于多肽而言)计算相似性和同一性，然后将结果排列成距离矩阵。序列相似性示于对角线下半部，而序列同一性示于对角线上半部。The global similarity and identity percentage between the full-length polypeptide sequences that can be used for implementing the method of the present invention, utilize one of methods available in the art namely MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics.20034: 29.MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella JJ, Bitincka L, Smalley J; software hosted by Ledion Bitincka) to determine. MatGAT software can generate similarity/identity matrices of DNA or protein sequences without pre-aligning the data. The program performs a series of pairwise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12 and a gap extension penalty of 2) and calculates similarity and identity using, for example, Blosum 62 (for polypeptides). , and then arrange the results into a distance matrix. Sequence similarity is shown on the lower diagonal, while sequence identity is shown on the upper diagonal.
比较所用的参数为：The parameters used for comparison are:
记分矩阵：Blosum 62Scoring Matrix: Blosum 62
首个空位：12First available slot: 12
延伸空位：2Extended Slots: 2
多肽序列全长范围的全局相似性和同一性的软件分析结果示于表B2。同一性百分比以粗体示于对角线上方，而相似性百分比示于对角线下方(正面)。The software analysis results of the global similarity and identity over the entire length of the polypeptide sequences are shown in Table B2. The percent identity is shown in bold above the diagonal, while the percent similarity is shown below the diagonal (front side).
与SEQ ID NO：102(拟南芥_AT3G33520.1)相比较，用于进行本发明方法的ARP6多肽序列之间的同一性百分比可低至50.8％氨基酸同一性。Compared with SEQ ID NO: 102 (Arabidopsis_AT3G33520.1), the percent identity between the ARP6 polypeptide sequences used to perform the methods of the invention can be as low as 50.8% amino acid identity.
表B2：多肽序列的全长范围的全局相似性和同一性的MatGAT结果。Table B2: MatGAT results for global similarity and identity over the full length range of polypeptide sequences.
3.6.POP多肽3.6. POP polypeptide
可以用于实施本发明方法的全长多肽序列之间的全局相似性和同一性百分比，利用本领域可用方法之一即MatGAT(矩阵全局比对工具)软件(BMC Bioinformatics.20034：29.MatGAT：an application thatgenerates similarity/identity matrices using protein or DNA sequences.Campanella JJ，Bitincka L，Smalley J；软件由Ledion Bitincka托管)来确定。MatGAT软件无需对数据进行预比对，即可产生DNA或蛋白质序列的相似性/同一性矩阵。该程序利用Myers和Miller全局比对算法(空位开放罚分为12，而空位延伸罚分为2)进行一系列的两两比对，利用例如Blosum 62(对于多肽而言)计算相似性和同一性，然后将结果排列成距离矩阵。序列相似性示于对角线下半部，而序列同一性示于对角线上半部。The global similarity and identity percentage between the full-length polypeptide sequences that can be used for implementing the method of the present invention, utilize one of methods available in the art namely MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics.20034: 29.MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella JJ, Bitincka L, Smalley J; software hosted by Ledion Bitincka) to determine. MatGAT software can generate similarity/identity matrices of DNA or protein sequences without pre-aligning the data. The program performs a series of pairwise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12 and a gap extension penalty of 2) and calculates similarity and identity using, for example, Blosum 62 (for polypeptides). , and then arrange the results into a distance matrix. Sequence similarity is shown on the lower diagonal, while sequence identity is shown on the upper diagonal.
比较所用的参数为：The parameters used for comparison are:
记分矩阵：Blosum 62Scoring Matrix: Blosum 62
首个空位：12First available slot: 12
延伸空位：2Extended Slots: 2
多肽序列全长范围的全局相似性和同一性的软件分析结果示于表B3。同一性百分比以粗体示于对角线上方，而相似性百分比示于对角线下方(正面)。The software analysis results of the global similarity and identity over the entire length of the polypeptide sequences are shown in Table B3. The percent identity is shown in bold above the diagonal, while the percent similarity is shown below the diagonal (front side).
与SEQ ID NO：117(At5g24260)相比较，用于进行本发明方法的POP多肽序列之间的同一性百分比可低至16.8％氨基酸同一性。Compared with SEQ ID NO: 117 (At5g24260), the percent identity between the POP polypeptide sequences used to perform the methods of the invention can be as low as 16.8% amino acid identity.
表B3：多肽序列的全长范围的全局相似性和同一性的MatGAT结果。Table B3: MatGAT results for global similarity and identity over the full length range of polypeptide sequences.
3.7.Crumpled Leaf (CRL)多肽3.7. Crumpled Leaf (CRL) peptides
可以用于实施本发明方法的全长多肽序列之间的全局相似性和同一性百分比，利用本领域可用方法之一即MatGAT(矩阵全局比对工具)软件(BMC Bioinformatics.2003 4：29.MatGAT：an application that generatessimilarity/identity matrices using protein or DNA sequences.CampanellaJJ，Bitincka L，Smalley J；软件由Ledion Bitincka托管)来确定。MatGAT软件无需对数据进行预比对，即可产生DNA或蛋白质序列的相似性/同一性矩阵。该程序利用Myers和Miller全局比对算法(空位开放罚分为12，而空位延伸罚分为2)进行一系列的两两比对，利用例如Blosum 62(对于多肽而言)计算相似性和同一性，然后将结果排列成距离矩阵。序列相似性示于对角线下半部，而序列同一性示于对角线上半部。The global similarity and identity percentage between the full-length polypeptide sequences that can be used for implementing the method of the present invention, utilize one of methods available in the art namely MatGAT (matrix global comparison tool) software (BMC Bioinformatics.2003 4: 29.MatGAT : an application that generates similarity/identity matrices using protein or DNA sequences. Campanella JJ, Bitincka L, Smalley J; software hosted by Ledion Bitincka) to determine. MatGAT software can generate similarity/identity matrices of DNA or protein sequences without pre-aligning the data. The program performs a series of pairwise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12 and a gap extension penalty of 2) and calculates similarity and identity using, for example, Blosum 62 (for polypeptides). , and then arrange the results into a distance matrix. Sequence similarity is shown on the lower diagonal, while sequence identity is shown on the upper diagonal.
比较所用的参数为：The parameters used for comparison are:
记分矩阵：Blosum 62Scoring Matrix: Blosum 62
首个空位：12First available slot: 12
延伸空位：2Extended Slots: 2
多肽序列全长范围的全局相似性和同一性的软件分析结果示于表B4。同一性百分比以粗体示于对角线上方，而相似性百分比示于对角线下方(正面)。The software analysis results of the global similarity and identity over the entire length of the polypeptide sequences are shown in Table B4. The percent identity is shown in bold above the diagonal, while the percent similarity is shown below the diagonal (front side).
与SEQ ID NO：156相比较，用于进行本发明方法的CRL多肽序列之间的同一性百分比可低至48％氨基酸同一性。Compared to SEQ ID NO: 156, the percent identity between the CRL polypeptide sequences used to perform the methods of the invention can be as low as 48% amino acid identity.
实施例4：鉴定可以用于实施本发明方法的多肽序列中包含的结构域Example 4: Identification of domains comprised in polypeptide sequences that can be used to practice the methods of the invention
4.1.Alfin样多肽4.1. Alfin-like peptides
蛋白质家族、结构域和位点整合资源(Integrated Resource of ProteinFamilies，Domains and Sites(InterPro))数据库是进行基于文本以及序列的搜索的、常用标签数据库的一个整合界面。InterPro数据库将这些数据库结合起来，这些数据库利用不同的方法学和有关充分表征的蛋白质的不同程度的生物信息来产生蛋白质标签。合作数据库包括SWISS-PROT、PROSITE、TrEMBL、PRINTS、ProDom和Pfam、Smart和TIGRFAMs。Pfam是覆盖许多常见蛋白质结构域和家族的、多重序列比对和隐马尔可夫模型的大集合。Pfam由位于英国的桑格研究所服务器(Sanger Instituteserver)托管。Interpro由位于英国的欧洲生物信息学研究所(EuropeanBioinfo rmatics Institute)托管。The Integrated Resource of Protein Families, Domains and Sites (InterPro) database is an integrated interface to commonly used label databases for text-based and sequence-based searches. InterPro databases combine these databases, which utilize different methodologies and varying degrees of biological information about well-characterized proteins to generate protein tags. Collaborative databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted on the Sanger Institute server in the UK. Interpro is hosted by the European Bioinfo rmatics Institute in the UK.
4.2.YRP多肽4.2. YRP polypeptide
蛋白质家族、结构域和位点整合资源(InterPro)数据库是进行基于文本以及序列的搜索的、常用标签数据库的一个整合界面。InterPro数据库将这些数据库结合起来，这些数据库利用不同的方法学和有关充分表征的蛋白质的不同程度的生物信息来产生蛋白质标签。合作数据库包括SWISS-PROT、PROSITE、TrEMBL、PRINTS、ProDom和Pfam、Smart和TIGRFAMs。Pfam是覆盖许多常见蛋白质结构域和家族的、多重序列比对和隐马尔可夫模型的大集合。Pfam由位于英国的桑格研究所服务器托管。Interpro由位于英国的欧洲生物信息学研究所托管。The Integrated Resource for Protein Families, Domains, and Sites (InterPro) database is an integrated interface to commonly used tag databases for text-based and sequence-based searches. InterPro databases combine these databases, which utilize different methodologies and varying degrees of biological information about well-characterized proteins to generate protein tags. Collaborative databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted on the servers of the Sanger Institute in the UK. Interpro is hosted by the European Bioinformatics Institute in the UK.
4.3.Brevis Radix样(BRXL)多肽4.3. Brevis Radix-like (BRXL) peptides
蛋白质家族、结构域和位点整合资源(InterPro)数据库是进行基于文本以及序列的搜索的、常用标签数据库的一个整合界面。InterPro数据库将这些数据库结合起来，这些数据库利用不同的方法学和有关充分表征的蛋白质的不同程度的生物信息来产生蛋白质标签。合作数据库包括SWISS-PROT、PROSITE、TrEMBL、PRINTS、ProDom和Pfam、Smart和TIGRFAMs。Interpro由位于英国的欧洲生物信息学研究所托管。The Integrated Resource for Protein Families, Domains, and Sites (InterPro) database is an integrated interface to commonly used tag databases for text-based and sequence-based searches. InterPro databases combine these databases, which utilize different methodologies and varying degrees of biological information about well-characterized proteins to generate protein tags. Collaborative databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Interpro is hosted by the European Bioinformatics Institute in the UK.
SEQ ID NO：18所示多肽序列的InterPro扫描结果示于表C1。The InterPro scanning results of the polypeptide sequence shown in SEQ ID NO: 18 are shown in Table C1.
表C1：SEQ ID NO：18所示多肽序列的InterPro扫描结果Table C1: InterPro scanning results of the polypeptide sequence shown in SEQ ID NO: 18
4.4.silky-1样多肽4.4. silky-1-like polypeptide
蛋白质家族、结构域和位点整合资源(InterPro)数据库是进行基于文本以及序列的搜索的、常用标签数据库的一个整合界面。InterPro数据库将这些数据库结合起来，这些数据库利用不同的方法学和有关充分表征的蛋白质的不同程度的生物信息来产生蛋白质标签。合作数据库包括SWISS-PROT、PROSITE、TrEMBL、PRINTS、ProDom和Pfam、Smart和TIGRFAMs。Pfam是覆盖许多常见蛋白质结构域和家族的、多重序列比对和隐马尔可夫模型的大集合。Pfam由位于英国的桑格研究所服务器托管。Interpro由位于英国的欧洲生物信息学研究所托管。The Integrated Resource for Protein Families, Domains, and Sites (InterPro) database is an integrated interface to commonly used tag databases for text-based and sequence-based searches. InterPro databases combine these databases, which utilize different methodologies and varying degrees of biological information about well-characterized proteins to generate protein tags. Collaborative databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted on the servers of the Sanger Institute in the UK. Interpro is hosted by the European Bioinformatics Institute in the UK.
4.5.ARP6多肽4.5. ARP6 polypeptide
蛋白质家族、结构域和位点整合资源(InterPro)数据库是进行基于文本以及序列的搜索的、常用标签数据库的一个整合界面。InterPro数据库将这些数据库结合起来，这些数据库利用不同的方法学和有关充分表征的蛋白质的不同程度的生物信息来产生蛋白质标签。合作数据库包括SWISS-PROT、PROSITE、TrEMBL、PRINTS、ProDom和Pfam、Smart和TIGRFAMs。Pfam是覆盖许多常见蛋白质结构域和家族的、多重序列比对和隐马尔可夫模型的大集合。Pfam由位于英国的桑格研究所服务器托管。Interpro由位于英国的欧洲生物信息学研究所托管。The Integrated Resource for Protein Families, Domains, and Sites (InterPro) database is an integrated interface to commonly used tag databases for text-based and sequence-based searches. InterPro databases combine these databases, which utilize different methodologies and varying degrees of biological information about well-characterized proteins to generate protein tags. Collaborative databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted on the servers of the Sanger Institute in the UK. Interpro is hosted by the European Bioinformatics Institute in the UK.
4.6.POP多肽4.6. POP polypeptide
蛋白质家族、结构域和位点整合资源(InterPro)数据库是进行基于文本以及序列的搜索的、常用标签数据库的一个整合界面。InterPro数据库将这些数据库结合起来，这些数据库利用不同的方法学和有关充分表征的蛋白质的不同程度的生物信息来产生蛋白质标签。合作数据库包括SWISS-PROT、PROSITE、TrEMBL、PRINTS、ProDom和Pfam、Smart和TIGRFAMs。Pfam是覆盖许多常见蛋白质结构域和家族的、多重序列比对和隐马尔可夫模型的大集合。Pfam由位于英国的桑格研究所服务器托管。Interpro由位于英国的欧洲生物信息学研究所托管。The Integrated Resource for Protein Families, Domains, and Sites (InterPro) database is an integrated interface to commonly used tag databases for text-based and sequence-based searches. InterPro databases combine these databases, which utilize different methodologies and varying degrees of biological information about well-characterized proteins to generate protein tags. Collaborative databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted on the servers of the Sanger Institute in the UK. Interpro is hosted by the European Bioinformatics Institute in the UK.
SEQ ID NO：117所示多肽序列的InterPro扫描结果示于表C2。The InterPro scanning results of the polypeptide sequence shown in SEQ ID NO: 117 are shown in Table C2.
表C2：SEQ ID NO：117所示多肽序列的InterPro扫描结果(主登录号)。Table C2: InterPro scanning results of the polypeptide sequence shown in SEQ ID NO: 117 (master accession number).
4.7.Crumpled Leaf(CRL)多肽4.7. Crumpled Leaf (CRL) peptide
蛋白质家族、结构域和位点整合资源(InterPro)数据库是进行基于文本以及序列的搜索的、常用标签数据库的一个整合界面。InterPro数据库将这些数据库结合起来，这些数据库利用不同的方法学和有关充分表征的蛋白质的不同程度的生物信息来产生蛋白质标签。合作数据库包括SWISS-PROT、PROSITE、TrEMBL、PRINTS、ProDom和Pfam、Smart和TIGRFAMs。Pfam是覆盖许多常见蛋白质结构域和家族的、多重序列比对和隐马尔可夫模型的大集合。Pfam由位于英国的桑格研究所服务器托管。Interpro由位于英国的欧洲生物信息学研究所托管。The Integrated Resource for Protein Families, Domains, and Sites (InterPro) database is an integrated interface to commonly used tag databases for text-based and sequence-based searches. InterPro databases combine these databases, which utilize different methodologies and varying degrees of biological information about well-characterized proteins to generate protein tags. Collaborative databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted on the servers of the Sanger Institute in the UK. Interpro is hosted by the European Bioinformatics Institute in the UK.
SEQ ID NO：156所示多肽序列的InterPro扫描结果示于表C3。The InterPro scanning results of the polypeptide sequence shown in SEQ ID NO: 156 are shown in Table C3.
表C3：SEQ ID NO：156所示多肽序列的InterPro扫描结果(主登录号)。Table C3: InterPro scanning results of the polypeptide sequence shown in SEQ ID NO: 156 (master accession number).
实施例5：用于实施本发明的方法的多肽序列的拓扑学预测Example 5: Topology Prediction of Polypeptide Sequences for Implementing the Methods of the Invention
5.1.Alfin样多肽5.1. Alfin-like peptides
TargetP 1.1预测真核蛋白质的亚细胞定位。位置分配所基于的是如下任一N-端前序列的预测性存在：叶绿体转运肽(cTP)、线粒体靶向肽(mTP)或分泌途径信号肽(SP)。最终预测所基于的分值并非真正的概率，且加起来并不必为1。不过，根据TargetP，得分最高的定位是最可能的，且分值之间的关系(可靠性级别)可作为所述预测的可靠性的指标。可靠性级别(RC)范围从1到5，其中1表示最强的预测。TargetP由丹麦技术大学(TechnicalUniversity of Denmark)的服务器维护。对于经预测包含N末端前序列的序列，还可预测潜在切割位点。TargetP 1.1 predicts the subcellular localization of eukaryotic proteins. Position assignments were based on the predicted presence of any of the following N-terminal prosequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP), or secretory pathway signal peptide (SP). The scores on which the final prediction is based are not true probabilities and do not necessarily add up to 1. However, according to TargetP, the location with the highest score is the most likely, and the relationship between the scores (reliability level) can be used as an indicator of the reliability of the prediction. The reliability level (RC) ranges from 1 to 5, with 1 indicating the strongest prediction. TargetP is maintained by servers at the Technical University of Denmark. For sequences predicted to contain an N-terminal presequence, potential cleavage sites can also be predicted.
可以选择许多参数，例如生物组别(非植物或植物)、截断值设置(无、预定的截断值设置、或用户指定的截断值设置)和预测切割位点的计算(是或否)。A number of parameters can be selected, such as organism group (non-plant or plant), cut-off setting (none, predetermined cut-off setting, or user-specified cut-off setting), and calculation of predicted cleavage sites (yes or no).
许多其他算法可用于实施此类分析，包括：Many other algorithms can be used to perform this type of analysis, including:
·在丹麦技术大学的服务器上托管的ChloroP 1.1；ChloroP 1.1 hosted on servers at the Technical University of Denmark;
·在澳大利亚布里斯班的昆士兰大学分子生物科学学院(Institute for Molecular Bioscience)的服务器上托管的ProteinProwler Subcellular Localisation Predictor 1.2版；· ProteinProwler Subcellular Localization Predictor version 1.2 hosted on servers at the Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
·在Edmonton，Alberta，Canada的阿尔伯特大学(Universityof Alberta)的服务器上托管的PENCE Proteome Analyst PA-GOSUB2.5；PENCE Proteome Analyst PA-GOSUB2.5 hosted on servers at the University of Alberta in Edmonton, Alberta, Canada;
·在丹麦技术大学的服务器上托管的TMHMM；· TMHMM hosted on servers at the Technical University of Denmark;
·PSORT(URL：psort.org)· PSORT (URL: psort.org)
·PLOC(Park和Kanehisa，Bioinformatics，19，1656-1663，2003)。• PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).
5.2.YRP多肽5.2. YRP polypeptide
TargetP 1.1预测真核蛋白质的亚细胞定位。位置分配所基于的是如下任一N-端前序列的预测性存在：叶绿体转运肽(cTP)、线粒体靶向肽(mTP)或分泌途径信号肽(SP)。最终预测所基于的分值并非真正的概率，且加起来并不必为1。不过，根据TargetP，得分最高的定位是最可能的，且分值之间的关系(可靠性级别)可作为所述预测的可靠性的指标。可靠性级别(RC)范围从1到5，其中1表示最强的预测。TargetP由丹麦技术大学的服务器维护。对于经预测包含N末端前序列的序列，还可预测潜在切割位点。TargetP 1.1 predicts the subcellular localization of eukaryotic proteins. Position assignments were based on the predicted presence of any of the following N-terminal prosequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP), or secretory pathway signal peptide (SP). The scores on which the final prediction is based are not true probabilities and do not necessarily add up to 1. However, according to TargetP, the location with the highest score is the most likely, and the relationship between the scores (reliability level) can be used as an indicator of the reliability of the prediction. The reliability level (RC) ranges from 1 to 5, with 1 indicating the strongest prediction. TargetP is maintained on servers at the Technical University of Denmark. For sequences predicted to contain an N-terminal presequence, potential cleavage sites can also be predicted.
可以选择许多参数，例如生物组别(非植物或植物)、截断值设置(无、预定的截断值设置、或用户指定的截断值设置)和预测切割位点的计算(是或否)。A number of parameters can be selected, such as organism group (non-plant or plant), cut-off setting (none, predetermined cut-off setting, or user-specified cut-off setting), and calculation of predicted cleavage sites (yes or no).
许多其他算法可用于实施此类分析，包括：Many other algorithms can be used to perform this type of analysis, including:
·在丹麦技术大学的服务器上托管的ChloroP 1.1；ChloroP 1.1 hosted on servers at the Technical University of Denmark;
·在澳大利亚布里斯班的昆士兰大学分子生物科学学院的服务器上托管的Protein Prowler Subcellular Localisation Predictor 1.2版；· Protein Prowler Subcellular Localization Predictor version 1.2 hosted on servers at the School of Molecular Biosciences, University of Queensland, Brisbane, Australia;
·在Edmonton，Alberta，Canada的阿尔伯特大学的服务器上托管的PENCE Proteome Analyst PA-GOSUB 2.5；PENCE Proteome Analyst PA-GOSUB 2.5 hosted on servers at the University of Alberta, Edmonton, Alberta, Canada;
·在丹麦技术大学的服务器上托管的TMHMM；· TMHMM hosted on servers at the Technical University of Denmark;
·PSORT(URL：psort.org)· PSORT (URL: psort.org)
·PLOC(Park和Kanehisa，Bioinformatics，19，1656-1663，2003)PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003)
5.3.silky-1样多肽5.3. silky-1-like polypeptide
TargetP 1.1预测真核蛋白质的亚细胞定位。位置分配所基于的是如下任一N-端前序列的预测性存在：叶绿体转运肽(cTP)、线粒体靶向肽(mTP)或分泌途径信号肽(SP)。最终预测所基于的分值并非真正的概率，且加起来并不必为1。不过，根据TargetP，得分最高的定位是最可能的，且分值之间的关系(可靠性级别)可作为所述预测的可靠性的指标。可靠性级别(RC)范围从1到5，其中1表示最强的预测。TargetP由丹麦技术大学的服务器维护。对于经预测包含N末端前序列的序列，还可预测潜在切割位点。TargetP 1.1 predicts the subcellular localization of eukaryotic proteins. Position assignments were based on the predicted presence of any of the following N-terminal prosequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP), or secretory pathway signal peptide (SP). The scores on which the final prediction is based are not true probabilities and do not necessarily add up to 1. However, according to TargetP, the location with the highest score is the most likely, and the relationship between the scores (reliability level) can be used as an indicator of the reliability of the prediction. The reliability level (RC) ranges from 1 to 5, with 1 indicating the strongest prediction. TargetP is maintained on servers at the Technical University of Denmark. For sequences predicted to contain an N-terminal presequence, potential cleavage sites can also be predicted.
可以选择许多参数，例如生物组别(非植物或植物)、截断值设置(无、预定的截断值设置、或用户指定的截断值设置)和预测切割位点的计算(是或否)。A number of parameters can be selected, such as organism group (non-plant or plant), cut-off setting (none, predetermined cut-off setting, or user-specified cut-off setting), and calculation of predicted cleavage sites (yes or no).
许多其他算法可用于实施此类分析，包括：Many other algorithms can be used to perform this type of analysis, including:
·在丹麦技术大学的服务器上托管的ChloroP 1.1；ChloroP 1.1 hosted on servers at the Technical University of Denmark;
·在澳大利亚布里斯班的昆士兰大学分子生物科学学院的服务器上托管的Protein Prowler Subcellular Localisation Predictor 1.2版；· Protein Prowler Subcellular Localization Predictor version 1.2 hosted on servers at the School of Molecular Biosciences, University of Queensland, Brisbane, Australia;
·在Edmonton，Alberta，Canada的阿尔伯特大学的服务器上托管的PENCE Proteome Analyst PA-GOSUB 2.5；PENCE Proteome Analyst PA-GOSUB 2.5 hosted on servers at the University of Alberta, Edmonton, Alberta, Canada;
·在丹麦技术大学的服务器上托管的TMHMM；· TMHMM hosted on servers at the Technical University of Denmark;
·PSORT(URL：psort.org)· PSORT (URL: psort.org)
·PLOC(Park和Kanehisa，Bioinformatics，19，1656-1663，2003)PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003)
5.4.ARP6多肽5.4. ARP6 polypeptide
TargetP 1.1预测真核蛋白质的亚细胞定位。位置分配所基于的是如下任一N-端前序列的预测性存在：叶绿体转运肽(cTP)、线粒体靶向肽(mTP)或分泌途径信号肽(SP)。最终预测所基于的分值并非真正的概率，且加起来并不必为1。不过，根据TargetP，得分最高的定位是最可能的，且分值之间的关系(可靠性级别)可作为所述预测的可靠性的指标。可靠性级别(RC)范围从1到5，其中1表示最强的预测。TargetP由丹麦技术大学的服务器维护。TargetP 1.1 predicts the subcellular localization of eukaryotic proteins. Position assignments were based on the predicted presence of any of the following N-terminal prosequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP), or secretory pathway signal peptide (SP). The scores on which the final prediction is based are not true probabilities and do not necessarily add up to 1. However, according to TargetP, the location with the highest score is the most likely, and the relationship between the scores (reliability level) can be used as an indicator of the reliability of the prediction. The reliability level (RC) ranges from 1 to 5, with 1 indicating the strongest prediction. TargetP is maintained on servers at the Technical University of Denmark.
对于经预测包含N末端前序列的序列，还可预测潜在切割位点。For sequences predicted to contain an N-terminal presequence, potential cleavage sites can also be predicted.
可以选择许多参数，例如生物组别(非植物或植物)、截断值设置(无、预定的截断值设置、或用户指定的截断值设置)和预测切割位点的计算(是或否)。A number of parameters can be selected, such as organism group (non-plant or plant), cut-off setting (none, predetermined cut-off setting, or user-specified cut-off setting), and calculation of predicted cleavage sites (yes or no).
许多其他算法可用于实施此类分析，包括：Many other algorithms can be used to perform this type of analysis, including:
·在丹麦技术大学的服务器上托管的ChloroP 1.1；ChloroP 1.1 hosted on servers at the Technical University of Denmark;
·在澳大利亚布里斯班的昆士兰大学分子生物科学学院的服务器上托管的Protein Prowler Subcellular Localisation Predictor 1.2版；· Protein Prowler Subcellular Localization Predictor version 1.2 hosted on servers at the School of Molecular Biosciences, University of Queensland, Brisbane, Australia;
·在Edmonton，Alberta，Canada的阿尔伯特大学的服务器上托管的PENCE Proteome Analyst PA-GOSUB 2.5；PENCE Proteome Analyst PA-GOSUB 2.5 hosted on servers at the University of Alberta, Edmonton, Alberta, Canada;
·在丹麦技术大学的服务器上托管的TMHMM；· TMHMM hosted on servers at the Technical University of Denmark;
·PSORT(URL：psort.org)· PSORT (URL: psort.org)
·PLOC(Park和Kanehisa，Bioinformatics，19，1656-1663，2003)PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003)
5.5.POP多肽5.5. POP polypeptide
TargetP 1.1预测真核蛋白质的亚细胞定位。位置分配所基于的是如下任一N-端前序列的预测性存在：叶绿体转运肽(cTP)、线粒体靶向肽(mTP)或分泌途径信号肽(SP)。最终预测所基于的分值并非真正的概率，且加起来并不必为1。不过，根据TargetP，得分最高的定位是最可能的，且分值之间的关系(可靠性级别)可作为所述预测的可靠性的指标。可靠性级别(RC)范围从1到5，其中1表示最强的预测。TargetP由丹麦技术大学的服务器维护。TargetP 1.1 predicts the subcellular localization of eukaryotic proteins. Position assignments were based on the predicted presence of any of the following N-terminal prosequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP), or secretory pathway signal peptide (SP). The scores on which the final prediction is based are not true probabilities and do not necessarily add up to 1. However, according to TargetP, the location with the highest score is the most likely, and the relationship between the scores (reliability level) can be used as an indicator of the reliability of the prediction. The reliability level (RC) ranges from 1 to 5, with 1 indicating the strongest prediction. TargetP is maintained on servers at the Technical University of Denmark.
对于经预测包含N末端前序列的序列，还可预测潜在切割位点。For sequences predicted to contain an N-terminal presequence, potential cleavage sites can also be predicted.
可以选择许多参数，例如生物组别(非植物或植物)、截断值设置(无、预定的截断值设置、或用户指定的截断值设置)和预测切割位点的计算(是或否)。A number of parameters can be selected, such as organism group (non-plant or plant), cut-off setting (none, predetermined cut-off setting, or user-specified cut-off setting), and calculation of predicted cleavage sites (yes or no).
SEQ ID NO：117所示的多肽序列的TargetP 1.1分析结果示于表D1。已选择“植物”生物组，无截断值定义，且转运肽的预测长度被要求。SEQ IDNO：117所示的多肽序列的亚细胞定位可能是细胞质或细胞核，预测无转运肽。The TargetP 1.1 analysis results of the polypeptide sequence shown in SEQ ID NO: 117 are shown in Table D1. The "Plant" organism group has been selected, no cutoff value has been defined, and the predicted length of the transit peptide is requested. The subcellular location of the polypeptide sequence shown in SEQ ID NO: 117 may be cytoplasm or nucleus, and no transit peptide is predicted.
表D1：SEQ ID NO：117所示的多肽序列的TargetP 1.1分析。缩写：Len，长度；cTP，叶绿体转运肽；mTP，线粒体转运肽；SP，分泌途径信号肽；其他，其他亚细胞靶向；Loc，预测的定位；RC，可靠性级别；Tplen，预测的转运肽长度。Table D1: TargetP 1.1 analysis of the polypeptide sequence shown in SEQ ID NO: 117. Abbreviations: Len, length; cTP, chloroplast transit peptide; mTP, mitochondrial transit peptide; SP, secretory pathway signal peptide; Other, other subcellular targeting; Loc, predicted localization; RC, reliability level; Tplen, predicted transport Peptide length.
许多其他算法可用于实施此类分析，包括：Many other algorithms can be used to perform this type of analysis, including:
·在丹麦技术大学的服务器上托管的ChloroP 1.1；ChloroP 1.1 hosted on servers at the Technical University of Denmark;
·在澳大利亚布里斯班的昆士兰大学分子生物科学学院的服务器上托管的Protein Prowler Subcellular Localisation Predictor 1.2版；· Protein Prowler Subcellular Localization Predictor version 1.2 hosted on servers at the School of Molecular Biosciences, University of Queensland, Brisbane, Australia;
·在Edmonton，Alberta，Canada的阿尔伯特大学的服务器上托管的PENCE Proteome Analyst PA-GOSUB 2.5；PENCE Proteome Analyst PA-GOSUB 2.5 hosted on servers at the University of Alberta, Edmonton, Alberta, Canada;
·在丹麦技术大学的服务器上托管的TMHMM；· TMHMM hosted on servers at the Technical University of Denmark;
·PSORT(URL：psort.org)· PSORT (URL: psort.org)
·PLOC(Park和Kanehisa，Bioinformatics，19，1656-1663，2003)。• PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).
5.6.Crumpled Leaf(CRL)多肽5.6. Crumpled Leaf (CRL) peptide
TargetP 1.1预测真核蛋白质的亚细胞定位。位置分配所基于的是如下任一N-端前序列的预测性存在：叶绿体转运肽(cTP)、线粒体靶向肽(mTP)或分泌途径信号肽(SP)。最终预测所基于的分值并非真正的概率，且加起来并不必为1。不过，根据TargetP，得分最高的定位是最可能的，且分值之间的关系(可靠性级别)可作为所述预测的可靠性的指标。可靠性级别(RC)范围从1到5，其中1表示最强的预测。TargetP由丹麦技术大学的服务器维护。TargetP 1.1 predicts the subcellular localization of eukaryotic proteins. Position assignments were based on the predicted presence of any of the following N-terminal prosequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP), or secretory pathway signal peptide (SP). The scores on which the final prediction is based are not true probabilities and do not necessarily add up to 1. However, according to TargetP, the location with the highest score is the most likely, and the relationship between the scores (reliability level) can be used as an indicator of the reliability of the prediction. The reliability level (RC) ranges from 1 to 5, with 1 indicating the strongest prediction. TargetP is maintained on servers at the Technical University of Denmark.
对于经预测包含N末端前序列的序列，还可预测潜在切割位点。For sequences predicted to contain an N-terminal presequence, potential cleavage sites can also be predicted.
可以选择许多参数，例如生物组别(非植物或植物)、截断值设置(无、预定的截断值设置、或用户指定的截断值设置)和预测切割位点的计算(是或否)。A number of parameters can be selected, such as organism group (non-plant or plant), cut-off setting (none, predetermined cut-off setting, or user-specified cut-off setting), and calculation of predicted cleavage sites (yes or no).
许多其他算法可用于实施此类分析，包括：Many other algorithms can be used to perform this type of analysis, including:
·在丹麦技术大学的服务器上托管的ChloroP 1.1；ChloroP 1.1 hosted on servers at the Technical University of Denmark;
·在澳大利亚布里斯班的昆士兰大学分子生物科学学院的服务器上托管的Protein Prowler Subcellular Localisation Predictor 1.2版；· Protein Prowler Subcellular Localization Predictor version 1.2 hosted on servers at the School of Molecular Biosciences, University of Queensland, Brisbane, Australia;
·在Edmonton，Alberta，Canada的阿尔伯特大学的服务器上托管的PENCE Proteome Analyst PA-GOSUB 2.5；PENCE Proteome Analyst PA-GOSUB 2.5 hosted on servers at the University of Alberta, Edmonton, Alberta, Canada;
·在丹麦技术大学的服务器上托管的TMHMM；· TMHMM hosted on servers at the Technical University of Denmark;
·PSORT(URL：psort.org)· PSORT (URL: psort.org)
·PLOC(Park和Kanehisa，Bioinformatics，19，1656-1663，2003)PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003)
使用一些上述算法获得的蛋白质分析结果示于下面：Protein analysis results obtained using some of the above algorithms are shown below:
Psort：内质网0.600Psort: endoplasmic reticulum 0.600
WOLFPsort：cyto：4.0，extr：3.0，E.R.：2.5，E.R._plas：2.5，plas：1.5，nucl：1.0，mito：1.0WOLFPsort: cyto: 4.0, extr: 3.0, E.R.: 2.5, E.R._plas: 2.5, plas: 1.5, nucl: 1.0, mito: 1.0
TargetP：chloro：0.775质量4TargetP:chloro:0.775
ChloroP：非叶绿体0.497ChloroP: non-chloroplast 0.497
MitoProtII：非线粒体0.027MitoProtII: non-mitochondrial 0.027
PTS1过氧化物酶体：未靶向的PTS1 peroxisomes: not targeted
Phobius：存在膜结构域[19-36]。Phobius: presence of a membrane domain [19-36].
实施例6：用于实施本发明方法的多肽序列的亚细胞定位预测Example 6: Prediction of Subcellular Location of Polypeptide Sequences for Implementing the Methods of the Invention
6.1.Brevis Radix样(BRXL)多肽6.1. Brevis Radix-like (BRXL) polypeptide
用于蛋白质定位的实验性方法范围广泛，从免疫定位到利用绿色荧光蛋白(GFP)或β-葡糖醛酸糖苷酶(GUS)标记蛋白质。鉴定BRXL多肽的亚细胞区室化的这类方法在本领域是公知的。Experimental methods for protein localization range from immunolocalization to labeling proteins with green fluorescent protein (GFP) or β-glucuronidase (GUS). Such methods for identifying subcellular compartmentalization of BRXL polypeptides are well known in the art.
从序列数据进行了蛋白质定位的计算预测。在瑞士生物信息学研究所托管的ExPASy蛋白质组学工具处可以得到一些本领域技术人员公知的算法，例如PSort、TargetP、ChloroP、LocTree、Predotar、LipoP、MITOPROT、PATS、PTS1、SignalP、TMHMM、TMpred等等。Computational predictions of protein localization were made from sequence data. Some algorithms known to those skilled in the art are available at the ExPASy proteomics tool hosted by the Swiss Bioinformatics Institute, such as PSort, TargetP, ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM, TMpred etc.
TargetP 1.1预测真核蛋白质的亚细胞定位。位置分配所基于的是如下任一N-端前序列的预测性存在：叶绿体转运肽(cTP)、线粒体靶向肽(mTP)或分泌途径信号肽(SP)。最终预测所基于的分值并非真正的概率，且加起来并不必为1。不过，根据TargetP，得分最高的定位是最可能的，且分值之间的关系(可靠性级别)可作为所述预测的可靠性的指标。可靠性级别(RC)范围从1到5，其中1表示最强的预测。TargetP由丹麦技术大学的服务器维护。TargetP 1.1 predicts the subcellular localization of eukaryotic proteins. Position assignments were based on the predicted presence of any of the following N-terminal prosequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP), or secretory pathway signal peptide (SP). The scores on which the final prediction is based are not true probabilities and do not necessarily add up to 1. However, according to TargetP, the location with the highest score is the most likely, and the relationship between the scores (reliability level) can be used as an indicator of the reliability of the prediction. The reliability level (RC) ranges from 1 to 5, with 1 indicating the strongest prediction. TargetP is maintained on servers at the Technical University of Denmark.
对于经预测包含N末端前序列的序列，还可预测潜在切割位点。For sequences predicted to contain an N-terminal presequence, potential cleavage sites can also be predicted.
选择了许多参数，例如生物组别(非植物或植物)、截断值设置(无、预定的截断值设置、或用户指定的截断值设置)和预测切割位点的计算(是或否)。A number of parameters are selected, such as organism group (non-plant or plant), cut-off setting (none, predetermined cut-off setting, or user-specified cut-off setting) and calculation of predicted cleavage sites (yes or no).
SEQ ID NO：117所示的多肽序列的TargetP 1.1分析结果示于下表。已选择“植物”生物组，无截断值定义。SEQ ID NO：117所示的多肽序列的预测的亚细胞定位不是叶绿体，不是线粒体、不是分泌途径，而最可能是细胞核的。The TargetP 1.1 analysis results of the polypeptide sequence shown in SEQ ID NO: 117 are shown in the table below. The "Plant" organism group has been selected, no cutoff defined. The predicted subcellular location of the polypeptide sequence shown in SEQ ID NO: 117 is not chloroplast, not mitochondria, not secretory pathway, but most likely nuclear.
表显示SEQ ID NO：117所示的多肽序列的TargetP 1.1分析The table shows the TargetP 1.1 analysis of the polypeptide sequence shown in SEQ ID NO: 117
实施例7：与用于实施本发明方法的多肽序列相关的测定试验Example 7: Determination tests related to polypeptide sequences used for carrying out the methods of the invention
7.1.Brevis Radix样(BRXL)多肽7.1. Brevis Radix-like (BRXL) polypeptide
用于本发明方法的BRXL多肽(至少其天然形式)通常，但不必，具有转录调节活性和与其他蛋白质相互作用的能力。DNA结合活性和蛋白质-蛋白质相互作用可使用本领域内公知的技术(例如Current Protocols inMolecular Biology，第1和2卷，Ausubel等(1994)，Current Protocols中)在体外或体内容易地测定。BRXL多肽包含保守BRX结构域，已在酵母双杂交实验中显示其介导蛋白质-蛋白质相互作用。BRXL BRXL以及还有PRAF样蛋白质家族的内部和/或之间的同二聚化和异二聚化在本领域内是已知的(Briggs等(2006)Plant Physiol 140：1307-1316；van Leeuwen等(2004)Trends Plant Sci 9：378-384)。BRXL polypeptides used in the methods of the invention (at least in their native form) typically, but not necessarily, possess transcriptional regulatory activity and the ability to interact with other proteins. DNA binding activity and protein-protein interactions can be readily determined in vitro or in vivo using techniques well known in the art (e.g., Current Protocols in Molecular Biology, Vol. 1 and 2, Ausubel et al. (1994), Current Protocols). BRXL polypeptides contain a conserved BRX domain that has been shown to mediate protein-protein interactions in yeast two-hybrid experiments. Homodimerization and heterodimerization within and/or between BRXL BRXL and also PRAF-like protein families are known in the art (Briggs et al. (2006) Plant Physiol 140:1307-1316; van Leeuwen et al. (2004) Trends Plant Sci 9:378-384).
7.2.POP多肽7.2. POP polypeptide
可如Bastos等(Biochem J.388：29-38，2005)所述，测量POP活性：POP活性通过测量通过酶底物N-Suc-Gly-Pro-Leu-Gly-Pro-AMC的水解而释放的AMC(7-酰氨基-4-甲基香豆素)的荧光来测定，其中Suc代表琥珀酰基。在100μl终体积中于含有20μM底物的反应缓冲液[25mM Hepes和5mM DTT(二硫苏糖醇)，pH 7.5]中测定POP。如Grellier等(J.Biol.Chem.276，47078-47086，2001)中所述，记录通过酶促反应释放的AMC的荧光。还可在相同的实验条件下使用不同的肽(N-Boc-Val-Leu-Lys-AMC、N-Boc-Leu-Lys-Arg-AMC、N-Cbz-Val-Lys-Met-AMC、N-Boc-Leu-Gly-Arg-AMC、N-Boc-Ile-Gly-Gly-Arg-AMC、N-Suc-Leu-Tyr-AMC、N-Suc-Ala-Ala-Ala-AMC、N-Boc-Val-Pro-Arg-AMC、N-Suc-Gly-Pro-AMC、N-Cbz-Gly-Gly-Arg-AMC、N-Suc-Ala-Ala-Pro-Phe-AMC、N-Cbz-Phe-Arg-AMC、H-Gly-Arg-AMC、H-Gly-Phe-AMC、Ala-Ala-Phe-AMC、L-Arg-AMC和L-Ala-AMC以及L-Lys-Ala-AMC，其中Boc和Cbz分别代表叔丁氧羰基和苄氧羰基)，测定POP活性。为了确定动力学参数，将重组(0.67ng)或天然(0.26ng)POP在反应缓冲液中与变化的N-Suc-Gly-Pro-Leu-Gly-Pro-AMC底物浓度(3.12-100μM)一起温育，如上所述测量AMC释放，使用Cornish-Bowden的方法，通过双曲线回归，确定Km和Vmax值。使用kcat＝Vmax/[E]0，计算kcat，其中[E]0表示活性酶浓度。通过将蛋白质与系列浓度的不可逆氯甲烷POP Tc80抑制剂一起温育，进行活性POP的定量，如Grellier等(2001)中所描述的。POP activity can be measured as described by Bastos et al. (Biochem J. 388:29-38, 2005): POP activity is measured by the release by hydrolysis of the enzyme substrate N-Suc-Gly-Pro-Leu-Gly-Pro-AMC The fluorescence of AMC (7-amido-4-methylcoumarin) is measured, wherein Suc represents succinyl. POP was determined in reaction buffer [25 mM Hepes and 5 mM DTT (dithiothreitol), pH 7.5] containing 20 μM substrate in a final volume of 100 μl. Fluorescence of AMCs released by enzymatic reactions was recorded as described in Grellier et al. (J. Biol. Chem. 276, 47078-47086, 2001). Different peptides (N-Boc-Val-Leu-Lys-AMC, N-Boc-Leu-Lys-Arg-AMC, N-Cbz-Val-Lys-Met-AMC, N-Cbz-Val-Lys-Met-AMC, -Boc-Leu-Gly-Arg-AMC, N-Boc-Ile-Gly-Gly-Arg-AMC, N-Suc-Leu-Tyr-AMC, N-Suc-Ala-Ala-Ala-AMC, N-Boc -Val-Pro-Arg-AMC, N-Suc-Gly-Pro-AMC, N-Cbz-Gly-Gly-Arg-AMC, N-Suc-Ala-Ala-Pro-Phe-AMC, N-Cbz-Phe - Arg-AMC, H-Gly-Arg-AMC, H-Gly-Phe-AMC, Ala-Ala-Phe-AMC, L-Arg-AMC and L-Ala-AMC and L-Lys-Ala-AMC, wherein Boc and Cbz represent tert-butoxycarbonyl and benzyloxycarbonyl, respectively), to measure the POP activity. To determine kinetic parameters, recombinant (0.67ng) or native (0.26ng) POP was mixed with varying N-Suc-Gly-Pro-Leu-Gly-Pro-AMC substrate concentrations (3.12-100 μM) in reaction buffer Following incubation, AMC release was measured as described above andKm andVmax values were determined by hyperbolic regression using the method of Cornish-Bowden. kcat was calculated using kcat =Vmax /[E]0 , where [E]0 represents the active enzyme concentration. Quantification of active POP was performed by incubating the protein with serial concentrations of an irreversible methyl chloride POP Tc80 inhibitor, as described in Grellier et al. (2001).
实施例8：用于本发明方法的核酸序列的克隆Embodiment 8: the cloning of the nucleic acid sequence that is used for the method of the present invention
8.1.Alfin样多肽8.1. Alfin-like peptides
使用cDNA文库(在pCMV Sport 6.0中；Invitrogen，Paisley，UK)作为模板，通过PCR扩增核酸序列。在标准条件下使用Hifi Taq DNA聚合酶，在50μl PCR mix中使用200ng模板进行PCR。引物包括用于Gateway重组的AttB位点。也使用标准方法纯化扩增的PCR片段。接着进行Gateway操作的第一步，即BP反应，在此期间PCR片段与pDONR201质粒体内重组以产生Gateway术语所称的“进入(entry)克隆”。作为
含有SEQ ID NO：1或SEQ ID NO：3的进入克隆随后与用于稻转化的Destination载体一起用于LR反应。此载体在T-DNA边界内包含如下功能性元件：植物可选择的标记；可筛选的标记表达盒；和旨在与已克隆到进入克隆中的目的核酸序列进行LR体内重组的Gateway盒。用于组成型表达的稻GOS2启动子(SEQ ID NO：7)位于此Gateway盒的上游。Entry clones containing SEQ ID NO: 1 or SEQ ID NO: 3 were then used in LR reactions with the Destination vector for rice transformation. This vector contains the following functional elements within the T-DNA borders: a plant selectable marker; a selectable marker expression cassette; and a Gateway cassette designed for LR in vivo recombination with the nucleic acid sequence of interest cloned into the entry clone. The rice GOS2 promoter (SEQ ID NO: 7) for constitutive expression is located upstream of this Gateway cassette.
在LR重组步骤之后，根据本领域公知的方法，将所产生的表达载体pGOS2::alfin-like(图1)转化进农杆菌菌株LBA4044。After the LR recombination step, the resulting expression vector pGOS2::alfin-like (Fig. 1) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
8.2.YRP多肽8.2. YRP polypeptide
使用cDNA文库(在pCMV Sport 6.0中；Invitrogen，Paisley，UK)作为模板，通过PCR扩增核酸序列。在标准条件下使用Hifi Taq DNA聚合酶，在50μl PCR mix中使用200ng模板进行PCR。引物包括用于Gateway重组的AttB位点。也使用标准方法纯化扩增的PCR片段。接着进行Gateway操作的第一步，即BP反应，在此期间PCR片段与pDONR201质粒体内重组以产生Gateway术语所称的“进入(entry)克隆”。作为
含有SEQ ID NO：10或SEQ ID NO：12的进入克隆随后与用于稻转化的Destination载体一起用于LR反应。此载体在T-DNA边界内包含如下功能性元件：植物可选择的标记；可筛选的标记表达盒；和旨在与已克隆到进入克隆中的目的核酸序列进行LR体内重组的Gateway盒。用于组成型表达的稻GOS2启动子(SEQ ID NO：16)位于此Gateway盒的上游。Entry clones containing SEQ ID NO: 10 or SEQ ID NO: 12 were then used in LR reactions with the Destination vector for rice transformation. This vector contains the following functional elements within the T-DNA borders: a plant selectable marker; a selectable marker expression cassette; and a Gateway cassette designed for LR in vivo recombination with the nucleic acid sequence of interest cloned into the entry clone. The rice GOS2 promoter (SEQ ID NO: 16) for constitutive expression is located upstream of this Gateway cassette.
在LR重组步骤之后，根据本领域公知的方法，将所产生的表达载体pGOS2::YRP(图2)转化进农杆菌菌株LBA4044。After the LR recombination step, the resulting expression vector pGOS2::YRP (Figure 2) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
8.3.Brevis Radix样(BRXL)多肽8.3. Brevis Radix-like (BRXL) peptides
除非另外指出，否则根据(Sambrook(2001)Molecular Cloning：alaboratory manual，第3版Cold Spring Harbor Laboratory Press，CSH，New York)或Ausubel等(1994)，Current Protocols in Molecular Biology，Current Protocols的第1和2卷中描述的标准方案，进行重组DNA技术。用于植物分子工作的标准材料和方法描述于由BIOS ScientificPublications Ltd(UK)和Blackwell Scientific Publications(UK)出版的R.D.D.Croy编著的Plant Molecular Biology Labfax(1993)中。Unless otherwise indicated, according to (Sambrook (2001) Molecular Cloning: laboratory manual, 3rd edition Cold Spring Harbor Laboratory Press, CSH, New York) or Ausubel et al. (1994), Current Protocols in Molecular Biology, pp. 1 and 1 of Current Protocols Standard protocols described in volume 2 for recombinant DNA techniques. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993), edited by R.D.D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
使用cDNA库(利用来自不同发育阶段的白杨植物的RNA构建的)作为模板，通过PCR扩增编码SEQ ID NO：18所示的BRXL多肽序列的白杨核酸序列。包括用于Gateway重组的AttB位点的下列引物用于PCR扩增：Using a cDNA library (constructed using RNA from poplar plants of different developmental stages) as a template, the poplar nucleic acid sequence encoding the BRXL polypeptide sequence shown in SEQ ID NO: 18 was amplified by PCR. The following primers including the AttB site for Gateway recombination were used for PCR amplification:
1)prm 11475(SEQ ID NO：88，有义)：1) prm 11475 (SEQ ID NO: 88, meaningful):
5’-ggggacaagtttgtacaaaaaagcaggcttaaacaatgtttacgtgcatagc-3’5'-ggggacaagtttgtacaaaaaagcaggcttaaacaatgtttacgtgcatagc-3'
2)prm11476(SEQ ID NO：89，反向，互补)：2) prm11476 (SEQ ID NO: 89, reverse, complementary):
5’-ggggaccactttgtacaagaaagctgggtgcaatttaggtcatgggaaat-3’5'-ggggaccactttgtacaagaaagctgggtgcaatttaggtcatgggaaat-3'
在标准条件下使用Hifi Taq DNA聚合酶进行PCR。还使用标准方法扩增和纯化了预期长度(包含attB位点)的PCR片段。接着进行Gateway操作的第一步，即BP反应，在此期间PCR片段与pDONR201质粒体内重组以产生Gateway术语所称的“进入克隆”。作为
含有SEQ ID NO：17的进入克隆随后与用于稻转化的Destination载体一起用于LR反应。此载体在T-DNA边界内包含如下功能性元件：植物可选择的标记；可筛选的标记表达盒；和旨在与已克隆到进入克隆中的目的核酸序列进行LR体内重组的Gateway盒。用于组成型表达的稻GOS2启动子(SEQ ID NO：87)位于此Gateway盒的上游。The entry clone containing SEQ ID NO: 17 was then used in LR reactions with the Destination vector for rice transformation. This vector contains the following functional elements within the T-DNA borders: a plant selectable marker; a selectable marker expression cassette; and a Gateway cassette designed for LR in vivo recombination with the nucleic acid sequence of interest cloned into the entry clone. The rice GOS2 promoter (SEQ ID NO: 87) for constitutive expression is located upstream of this Gateway cassette.
在LR重组步骤之后，根据本领域公知的方法，将所产生的表达载体pGOS2::BRXL(图6)转化进农杆菌菌株LBA4044。After the LR recombination step, the resulting expression vector pGOS2::BRXL (Figure 6) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
8.4.silky-1样多肽8.4. silky-1-like polypeptide
使用cDNA文库(在pCMV Sport 6.0中；Invitrogen，Paisley，UK)作为模板，通过PCR扩增核酸序列。在标准条件下使用Hifi Taq DNA聚合酶，在50μl PCR mix中使用200ng模板进行PCR。引物包括用于Gateway重组的AttB位点。也使用标准方法纯化扩增的PCR片段。接着进行Gateway操作的第一步，即BP反应，在此期间PCR片段与pDONR201质粒体内重组以产生Gateway术语所称的“进入(entry)克隆”。作为
含有SEQ ID NO：90、SEQ ID NO：92或SEQ ID NO：94的进入克隆随后与用于稻转化的Destination载体一起用于LR反应。此载体在T-DNA边界内包含如下功能性元件：植物可选择的标记；可筛选的标记表达盒；和旨在与已克隆到进入克隆中的目的核酸序列进行LR体内重组的Gateway盒。用于组成型表达的稻GOS2启动子(SEQ ID NO：96)位于此Gateway盒的上游。Entry clones containing SEQ ID NO:90, SEQ ID NO:92 or SEQ ID NO:94 were then used in LR reactions with the Destination vector for rice transformation. This vector contains the following functional elements within the T-DNA borders: a plant selectable marker; a selectable marker expression cassette; and a Gateway cassette designed for LR in vivo recombination with the nucleic acid sequence of interest cloned into the entry clone. The rice GOS2 promoter (SEQ ID NO: 96) for constitutive expression is located upstream of this Gateway cassette.
在LR重组步骤之后，根据本领域公知的方法，将所产生的表达载体pGOS2::silky-1-like(图7)转化进农杆菌菌株LBA4044。After the LR recombination step, the resulting expression vector pGOS2::silky-1-like (Figure 7) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
8.5.ARP6多肽8.5. ARP6 polypeptide
使用定制的拟南芥幼苗cDNA文库(在pCMV Sport 6.0中；Invitrogen，Paisley，UK)作为模板，通过PCR扩增用于本发明方法中的核酸序列。在标准条件下使用Hifi Taq DNA聚合酶，在50μl PCR mix中使用200ng模板进行PCR。所使用的引物是：(SEQ ID NO：113；有义，起始密码子以粗体显示)：5’-ggggacaagtttgtacaaaaaagcaggcttaaacaatgtc aaacatcgttgttcta-3’和(SEQ ID NO：114；反向，互补)：5’-ggggaccact ttgtacaagaaagctgggtt catgtgatattgttttggtt-3’，其包括用于Gateway重组的AttB位点。也使用标准方法纯化了扩增的PCR片段。接着进行Gateway操作的第一步，即BP反应，在此期间PCR片段与pDONR201质粒体内重组以产生Gateway术语所称的“进入克隆”pARP6。作为
含有SEQ ID NO：101的进入克隆随后与用于稻转化的Destination载体一起用于LR反应。此载体在T-DNA边界内包含如下功能性元件：植物可选择的标记；可筛选的标记表达盒；和旨在与已克隆到进入克隆中的目的核酸序列进行LR体内重组的Gateway盒。用于组成型特异性表达的稻GOS2启动子(SEQ ID NO：115)位于此Gateway盒的上游。The entry clone containing SEQ ID NO: 101 was then used in LR reactions with the Destination vector for rice transformation. This vector contains the following functional elements within the T-DNA borders: a plant selectable marker; a selectable marker expression cassette; and a Gateway cassette designed for LR in vivo recombination with the nucleic acid sequence of interest cloned into the entry clone. The rice GOS2 promoter (SEQ ID NO: 115) for constitutive specific expression is located upstream of this Gateway cassette.
在LR重组步骤之后，根据本领域公知的方法，将所产生的表达载体pGOS2::ARP6(图10)转化进农杆菌菌株LBA4044。After the LR recombination step, the resulting expression vector pGOS2::ARP6 (Figure 10) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
8.6.POP多肽8.6. POP polypeptide
使用定制的拟南芥幼苗cDNA文库(在pCMV Sport 6.0中；Invitrogen，Paisley，UK)作为模板，通过PCR扩增在本发明方法中使用的核酸序列。在标准条件下使用Hifi Taq DNA聚合酶，在50μl PCR mix中使用200ng模板进行PCR。所使用的引物是：prm05611(SEQ ID NO：132；有义，起始密码子以粗体显示)：5’-ggggacaagtttgtacaaaaaagcaggcttaaacaatggcggataaggacgtt-3’和prm05612(SEQ ID NO：133；反向，互补)：5’-ggggaccactttgtacaagaaagctgggtaagcaacaacaggttctg tga-3’，其包括用于Gateway重组的AttB位点。也使用标准方法纯化了扩增的PCR片段。接着进行Gateway操作的第一步，即BP反应，在此期间PCR片段与pDONR201质粒体内重组以产生Gateway术语所称的“进入克隆”pPOP。作为
含有SEQ ID NO：116的进入克隆随后与用于稻转化的Destination载体一起用于LR反应。此载体在T-DNA边界内包含如下功能性元件：植物可选择的标记；可筛选的标记表达盒；和旨在与已克隆到进入克隆中的目的核酸序列进行LR体内重组的Gateway盒。用于组成型特异性表达的稻GOS2启动子(SEQ ID NO：131)位于此Gateway盒的上游。The entry clone containing SEQ ID NO: 116 was then used in LR reactions with the Destination vector for rice transformation. This vector contains the following functional elements within the T-DNA borders: a plant selectable marker; a selectable marker expression cassette; and a Gateway cassette designed for LR in vivo recombination with the nucleic acid sequence of interest cloned into the entry clone. The rice GOS2 promoter (SEQ ID NO: 131 ) for constitutive specific expression is located upstream of this Gateway cassette.
在LR重组步骤之后，根据本领域公知的方法，将所产生的表达载体pGOS2::POP(图13)转化进农杆菌菌株LBA4044。After the LR recombination step, the resulting expression vector pGOS2::POP (Figure 13) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
8.7.Crumpled Leaf (CRL)多肽8.7. Crumpled Leaf (CRL) Peptides
使用定制的拟南芥幼苗cDNA文库(在pCMV Sport 6.0中；Invitrogen，Paisley，UK)作为模板，通过PCR扩增在本发明方法中使用的核酸序列。在标准条件下使用Hifi Taq DNA聚合酶，在50μl PCR mix中使用200ng模板进行PCR。所使用的引物是：引物(SEQ ID NO：211；有义，起始密码子以粗体显示)：5’-ggggacaagtttgtacaaaaaagcaggcttaaacaatgggtaccgagtcggg-3’和引物(SEQ ID NO：212所示；反向，互补)：5’-ggggaccactttgtacaagaaagctgggt tcagacaatagaaaagggggt-3’，其包括用于Gateway重组的AttB位点。也使用标准方法纯化了扩增的PCR片段。接着进行Gateway操作的第一步，即BP反应，在此期间PCR片段与pDONR201质粒体内重组以产生Gateway术语所称的“进入克隆”pCRL。作为
含有SEQ ID NO：155的进入克隆随后与用于稻转化的Destination载体一起用于LR反应。此载体在T-DNA边界内包含如下功能性元件：植物可选择的标记；可筛选的标记表达盒；和旨在与已克隆到进入克隆中的目的核酸序列进行LR体内重组的Gateway盒。用于组成型特异性表达的稻GOS2启动子(SEQ ID NO：213)位于此Gateway盒的上游。The entry clone containing SEQ ID NO: 155 was then used in LR reactions with the Destination vector for rice transformation. This vector contains the following functional elements within the T-DNA borders: a plant selectable marker; a selectable marker expression cassette; and a Gateway cassette designed for LR in vivo recombination with the nucleic acid sequence of interest cloned into the entry clone. The rice GOS2 promoter (SEQ ID NO: 213) for constitutive specific expression is located upstream of this Gateway cassette.
在LR重组步骤之后，根据本领域公知的方法，将所产生的表达载体pGOS2::CRL(图3)转化进农杆菌菌株LBA4044。After the LR recombination step, the resulting expression vector pGOS2::CRL (Figure 3) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
实施例9：植物转化Example 9: Plant Transformation
稻转化rice transformation
用含表达载体的农杆菌转化稻(Oryza sativa)植物。使梗稻栽培种日本晴(Nipponbare)的成熟干种子脱壳。通过在70％乙醇中孵育1分钟，接着在0.2％HgCl2中孵育30分钟，接着用无菌蒸馏水洗6次，每次15分钟进行消毒。然后使消毒的种子在含有2，4-D的培养基(愈伤组织诱导培养基)上萌发。在黑暗中孵育四周之后，切下盾片来源的胚发生愈伤组织，并在相同的培养基中增殖。两周之后，通过在相同培养基中传代培养另外2周来扩增或者增殖愈伤组织。在共培养之前3天，在新鲜培养基上传代培养胚发生愈伤组织块(以加强细胞分裂活性)。Oryza sativa plants were transformed with Agrobacterium containing the expression vector. Mature dry seeds of the stalk rice cultivar Nipponbare were dehulled. Sterilize by incubating in 70% ethanol for 1 min, followed by 0.2%HgCl for 30 min, followed by 6 washes with sterile distilled water for 15 min each. The sterilized seeds were then germinated on medium containing 2,4-D (callus induction medium). After four weeks of incubation in the dark, scutellum-derived embryogenic calli were excised and propagated in the same medium. After two weeks, the calli were expanded or multiplied by subculturing in the same medium for another 2 weeks. Three days prior to co-cultivation, embryogenic callus pieces were subcultured on fresh medium (to enhance cell division activity).
含有表达载体的农杆菌菌株LBA4404用于共培养。农杆菌接种于含有合适抗生素的AB培养基上，并在28℃培养3天。接着收集细菌并悬浮在液体共培养培养基中至光密度(OD600)约为1。接着将悬浮液转移至培养皿，并将愈伤组织浸于悬浮液中15分钟。随后将愈伤组织在滤纸上沾干，转移至固化的共培养培养基中，并在黑暗中于25℃孵育3天。在选择剂的存在下，共培养的愈伤组织在含有2，4-D的培养基上于28℃暗培养四周。在此期间，发育出快速生长的抗性愈伤组织岛。将此材料转移至再生培养基并在光照下孵育之后，释放了胚发生潜力，在接下来的四至五周发育出芽。将芽从愈伤组织切下，并在含生长素的培养基中孵育2到3周，将其从培养基转移至土壤。变硬的芽在高湿度和短白昼条件下在温室中培养。Agrobacterium strain LBA4404 containing the expression vector was used for co-cultivation. Agrobacterium was inoculated on AB medium containing appropriate antibiotics and cultured at 28°C for 3 days. Bacteria were then collected and suspended in liquid co-cultivation medium to an optical density (OD600) of approximately 1. The suspension was then transferred to a Petri dish, and the calli were immersed in the suspension for 15 minutes. The calli were then blotted dry on filter paper, transferred to solidified co-cultivation medium, and incubated at 25°C in the dark for 3 days. Co-cultured calli were cultured in the dark at 28°C for four weeks on 2,4-D-containing medium in the presence of the selection agent. During this period, rapidly growing islands of resistant callus develop. After transferring this material to regeneration medium and incubating it in the light, the embryogenic potential was released and buds developed over the next four to five weeks. Shoots are excised from the callus and incubated for 2 to 3 weeks in an auxin-containing medium, from which they are transferred to soil. The hardened shoots are grown in greenhouses under conditions of high humidity and short days.
一个构建体产生约35个独立的T0稻转化体。将原代转化体从组织培养室转移到温室。在定量PCR分析验证T-DNA插入物的拷贝数后，只保留对选择剂表现出耐受性的单拷贝转基因植物用以收获T1种子。在移植后三至五个月收获种子。参见Aldemita和Hodges 1996，Chan等，1993，Hiei等，1994。One construct produced about 35 independent TO rice transformants. Transfer the primary transformants from the tissue culture room to the greenhouse. After quantitative PCR analysis to verify the copy number of the T-DNA insert, only single-copy transgenic plants exhibiting tolerance to the selection agent were kept for harvesting T1 seeds. Harvest the seeds three to five months after transplanting. See Aldemita and Hodges 1996, Chan et al., 1993, Hiei et al., 1994.
玉米转化corn transformation
用Ishida等(1996)Nature Biotech 14(6)：745-50所述方法的改良方案进行玉米(玉蜀黍)转化。在玉米中转化是基因型依赖性的，并且只有特定的基因型适于转化和再生。近交系A188(明尼苏达大学)或以A188为亲本的杂种是转化供体材料的优良来源，但是也可以成功使用其它基因型。授粉后约11天(DAP)，当未成熟胚的长度是约1至1.2mm时，从玉米植物收获穗。共培养未成熟胚和含有表达载体的根癌农杆菌，并通过器官发生回收转基因植物。切离的胚依次生长在含有选择剂(例如咪唑啉酮，但可使用多种选择标记)的愈伤组织诱导培养基、和玉米再生培养基上。培养板在光照下于25℃孵育2-3周，或直到芽发育。从每个胚中将绿芽转移到玉米生根培养基上并在25℃孵育2-3周，直到根发育。将生根的芽移植到温室的土壤中。从表现出对选择剂具有耐受性且含有单拷贝T-DNA插入片段的植物产生T1种子。Maize (maize) transformation was performed using a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation in maize is genotype dependent and only certain genotypes are suitable for transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are excellent sources of donor material for transformation, but other genotypes can also be used successfully. Ears are harvested from maize plants approximately 11 days after pollination (DAP), when immature embryos are approximately 1 to 1.2 mm in length. Immature embryos were co-cultured with Agrobacterium tumefaciens harboring the expression vector, and transgenic plants were recovered through organogenesis. Excised embryos are sequentially grown on callus induction medium containing a selection agent (eg imidazolinone, but a variety of selection markers can be used), followed by maize regeneration medium. Plates were incubated in the light at 25°C for 2-3 weeks, or until shoots developed. Green shoots from each embryo were transferred to maize rooting medium and incubated at 25°C for 2-3 weeks until roots developed. Rooted shoots are transplanted into soil in the greenhouse. T1 seeds were generated from plants exhibiting tolerance to the selection agent and containing a single copy of the T-DNA insert.
小麦转化wheat transformation
运用Ishida等(1996)Nature Biotech 14(6)：745-50描述的方法，进行小麦的转化。栽培种Bobwhite(可从CIMMYT，Mexico(墨西哥)获得)常用来进行转化。共培养未成熟胚和含有表达载体的根癌农杆菌，并通过器官发生回收转基因植株。与农杆菌孵育后，胚依次体外生长在含有选择试剂(例如咪唑啉酮，但可使用多种选择标记)的愈伤组织诱导培养基，和再生培养基上。培养板在光照下于25℃孵育2-3周，或直到芽发育。从每个胚中将绿芽转移到生根培养基上并在25℃孵育2-3周，直到根发育。将生根的芽移植到温室的土壤中。从表现出对选择剂具有耐受性且含有单拷贝T-DNA插入片段的植物产生T1种子。Transformation of wheat was performed using the method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used for transformation. Immature embryos were co-cultured with Agrobacterium tumefaciens containing the expression vector, and transgenic plants were recovered through organogenesis. Following incubation with Agrobacterium, the embryos are grown in vitro sequentially on callus induction medium containing a selection agent (eg imidazolinone, but a variety of selection markers can be used), followed by regeneration medium. Plates were incubated in the light at 25°C for 2-3 weeks, or until shoots developed. Green shoots from each embryo were transferred onto rooting medium and incubated at 25°C for 2-3 weeks until roots developed. Rooted shoots are transplanted into soil in the greenhouse. T1 seeds were generated from plants exhibiting tolerance to the selection agent and containing a single copy of the T-DNA insert.
大豆转化soybean conversion
根据Texas A&M专利US 5,164,310所述方法的改良方案转化大豆。若干商业大豆品种可以通过该方法转化。栽培种Jack(可以得自伊利诺斯种子公司(the Illinois Seed foundation))常用来进行转化。对大豆种子消毒以进行体外播种。从七日龄幼苗中切出下胚轴、胚根和一个子叶。进一步培养上胚轴和剩下的子叶以发育腋结。切离这些腋结并与含有表达载体的根癌农杆菌孵育。在共培养处理后，洗涤外植体并转移到选择培养基中。切离再生的芽，置于芽伸长培养基中。将长度不超过1cm的芽置于生根培养基中直到发育出根。将生根的芽移植到温室的土壤中。从对选择剂表现出耐受性且含有单拷贝T-DNA插入片段的植物产生T1种子。Soybean was transformed according to a modification of the method described in Texas A&M patent US 5,164,310. Several commercial soybean varieties can be transformed by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Disinfection of soybean seeds for in vitro sowing. Hypocotyls, radicles, and one cotyledon were excised from seven-day-old seedlings. The epicotyls and remaining cotyledons were further cultured to develop axillary nodes. These axillary nodes were excised and incubated with Agrobacterium tumefaciens containing the expression vector. After co-cultivation treatment, explants were washed and transferred to selection medium. Regenerated shoots were excised and placed on shoot elongation medium. Shoots up to 1 cm in length are placed on rooting medium until roots develop. Rooted shoots are transplanted into soil in the greenhouse. T1 seeds are generated from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
油菜籽/芸苔转化Rapeseed/Canola Transformation
利用5-6日龄幼苗的子叶柄和下胚轴作为外植体进行组织培养并根据Babic等(1998，Plant Cell Rep 17：183-188)进行转化。商业栽培种Westar(加拿大农业(Agriculture Canada))是用作转化的标准品种，但是也可以使用其它品种。对芸苔种子表面消毒以进行体外播种。从体外幼苗中切离附着有子叶的子叶柄外植体，并通过将子叶柄外植体的切割端浸入细菌悬浮液中来接种农杆菌(含有表达载体)。随后外植体在含有3mg/l BAP、3％蔗糖、0.7％植物琼脂(Phytagar)的MSBAP-3培养基中于23℃、16小时光照培养2天。与农杆菌共培养2天后，将子叶柄外植体转移到含有3mg/l BAP、头孢噻肟、羧苄青霉素或特美汀(300mg/l)的MSBAP-3培养基中7天，然后在含有头孢噻肟、羧苄青霉素或特美汀和选择剂的MSBAP-3培养基上培养直到芽再生。当芽长5-10mm时，将其切下并转移到芽伸长培养基(MSBAP-0.5，含有0.5mg/l BAP)中。将约2cm长的芽转移到生根培养基(MS0)中进行根诱导。将生根的芽移植到温室的土壤中。从对选择剂表现出耐受性且含有单拷贝T-DNA插入片段的植物产生T1种子。The cotyledon petioles and hypocotyls of 5-6 day old seedlings were used as explants for tissue culture and transformation according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but others may also be used. Surface disinfection of canola seeds for in vitro sowing. The cotyledonous petiole explant with the cotyledon attached was excised from the in vitro seedling, and Agrobacterium (containing the expression vector) was inoculated by dipping the cut end of the cotyledonous petiole explant into the bacterial suspension. Then the explants were cultured at 23° C. under 16 hours light for 2 days in MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, and 0.7% Phytagar. After 2 days of co-cultivation with Agrobacterium, the petiole explants were transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin or timentin (300 mg/l) for 7 days, and then in Culture on MSBAP-3 medium containing cefotaxime, carbenicillin or timentin and selection agent until shoot regeneration. When shoots were 5-10 mm long, they were excised and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots approximately 2 cm long were transferred to rooting medium (MS0) for root induction. Rooted shoots are transplanted into soil in the greenhouse. T1 seeds are generated from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
苜蓿转化alfalfa transformation
利用(McKersie等1999Plant Physiol 119：839-847)的方法转化苜蓿(紫花苜蓿(Medicago sativa))的再生克隆。苜蓿的再生和转化是基因型依赖性的，因此需要再生植株。获得再生植株的方法已有描述。例如，这些可以选自栽培种Rangelander(加拿大农业(Agriculture Canada))或如BrownDCW和A Atanassov(1985.Plant Cell Tissue Organ Culture 4：111-112)所述的任何其它商业苜蓿品种。可选的，选择RA3品种(威斯康辛大学(University of Wisconsin))用于组织培养(Walker等，1978 Am J Bot65：654-659)。子叶柄外植体与含有表达载体的根癌农杆菌C58C1 pMP90(McKersie等，1999 Plant Physiol 119：839-847)或LBA4404的过夜培养物进行共培养。外植体在含有288mg/L Pro、53mg/L硫代脯氨酸、4.35g/LK2SO4和100μm乙酰丁香酮的SH诱导培养基上在黑暗中共培养3天。外植体在半强度Murashige-Skoog培养基(Murashige和Skoog，1962)中洗涤，并置于相同的SH诱导培养基中，但该培养基不含乙酰丁香酮而含有合适的选择剂和合适的抗生素以抑制农杆菌生长。数周后，体细胞胚转移到不含生长调节剂、不含抗生素、含有50g/L蔗糖的BOi2Y发育培养基中。体细胞胚随后在半强度Murashige-Skoog培养基上萌发。生根的幼苗移植到花盆中并在温室中生长。从对选择剂表现出耐受性且含有单拷贝T-DNA插入片段的植物产生T1种子。Regenerating clones of alfalfa (Medicago sativa) were transformed using the method of (McKersie et al. 1999 Plant Physiol 119:839-847). Regeneration and transformation of alfalfa are genotype-dependent and thus require regenerated plants. Methods for obtaining regenerated plants have been described. For example, these may be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown DCW and A Atanassov (1985. Plant Cell Tissue Organ Culture 4:111-112). Alternatively, the RA3 variety (University of Wisconsin) was selected for tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants were co-cultured with overnight cultures of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. Explants were co-cultured in the dark for 3 days on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μM acetosyringone. Explants were washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and placed in the same SH-inducing medium, but without acetosyringone and containing a suitable selection agent and a suitable Antibiotics to inhibit the growth of Agrobacterium. Several weeks later, somatic embryos were transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos were subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings are transplanted into pots and grown in a greenhouse. T1 seeds are generated from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
棉花转化cotton transformation
按照US 5,159,135中描述的方法使用根癌农杆菌转化棉花。于3％次氯酸钠溶液中20分钟，对棉花种子表面消毒，并且在具有500μg/ml头孢噻肟的蒸馏水中进行洗涤。然后将种子转移至具有50μg/ml苯菌灵(benomyl)的SH培养基中进行萌发。从4至6日龄的幼苗中取出下胚轴，切成0.5厘米的小块，置于0.8％琼脂上。将农杆菌悬浮液(每ml大约108个细胞，从用目的基因和适当的选择标记转化的过夜培养物稀释的)用于接种下胚轴外植体。在室温和光照下3天后，将组织转移至具有Murashige和Skoog盐和B5维生素(Gamborg等，Exp.Cell Res.50：151-158(1968))、0.1mg/l2，4-D、0.1mg/l 6-糠氨基嘌呤(6-furfurylaminopurine)和750μg/mlMgCL2、以及50至100μg/ml头孢噻肟和400-500μg/ml羧苄青霉素(以杀死残留细菌)的固体培养基(1.6g/l Gelrite)。在2至3个月(每4至6周进行一次传代培养)后分离单细胞系并且将其在选择培养基上进一步培养以进行组织扩增(30℃，16小时光周期)。接着将转化的组织在非选择培养基上进一步培养2至3个月以产生体细胞胚。将至少4mm长的健康外貌的胚转移至具有SH培养基(于细小蛭石中)的试管中，所述培养基补充有0.1mg/l吲哚乙酸、6-糠氨基嘌呤和赤霉酸。将胚在30℃和16小时的光周期下进行培养，将2至3叶期的小植株转移入具有蛭石和营养物的花盆。植物变硬，然后转移至温室以进一步栽培。Cotton was transformed with Agrobacterium tumefaciens as described in US 5,159,135. Cotton seeds were surface sterilized in 3% sodium hypochlorite solution for 20 minutes and washed in distilled water with 500 μg/ml cefotaxime. Seeds were then transferred to SH medium with 50 μg/ml benomyl for germination. Hypocotyls were removed from 4- to 6-day-old seedlings, cut into 0.5 cm pieces, and placed on 0.8% agar. Agrobacterium suspensions (approximately 108 cells per ml, diluted from overnight cultures transformed with the gene of interest and the appropriate selectable marker) were used to inoculate hypocotyl explants. After 3 days at room temperature and in the light, the tissue was transferred to cells with Murashige and Skoog salts and B5 vitamins (Gamborg et al., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/l2, 4-D, 0.1 mg /l 6-furfurylaminopurine (6-furfurylaminopurine) and 750μg/ml MgCL2, and 50 to 100μg/ml cefotaxime and 400-500μg/ml carbenicillin (to kill residual bacteria) solid medium (1.6g/ l Gelrite). Single cell lines were isolated after 2 to 3 months (subcultured every 4 to 6 weeks) and further cultured on selective medium for tissue expansion (30°C, 16 hr photoperiod). The transformed tissue is then further cultured on non-selective medium for 2 to 3 months to produce somatic embryos. Healthy-appearing embryos at least 4 mm long were transferred to tubes with SH medium (in fine vermiculite) supplemented with 0.1 mg/l indoleacetic acid, 6-furfurylaminopurine and gibberellic acid. Embryos were cultured at 30°C with a 16-hour photoperiod, and plantlets at the 2-3 leaf stage were transferred into pots with vermiculite and nutrients. The plants harden and are then transferred to the greenhouse for further cultivation.
实施例10：表型评估方法Example 10: Phenotypic Assessment Methods
10.1评估设置10.1 Evaluation Setup
产生大约35个独立的T0稻转化体。原代转化体由组织培养室转移到温室进行生长并收获T1种子。保留其中T1代发生转基因的存在/缺乏的3∶1分离的6个事件。对于每一个此类事件，通过监测可视标记的表达，选出大约10个含转基因(杂合子和纯合子)的T1幼苗、以及大约10个缺少转基因(无效合子)的T1幼苗。转基因植物和相应的无效合子在随机位置上并排生长。温室条件为短白昼(12小时光照)，日间28℃，夜间22℃，相对湿度70％。Approximately 35 independent TO rice transformants were generated. Primary transformants were transferred from the tissue culture room to the greenhouse for growth and T1 seeds were harvested. Six events in which the T1 generation segregated 3:1 for the presence/absence of the transgene were retained. For each such event, approximately 10 T1 seedlings containing the transgene (heterozygous and homozygous) and approximately 10 T1 seedlings lacking the transgene (nullzygote) were selected by monitoring the expression of the visible marker. Transgenic plants and corresponding nullzygotes were grown side by side in random positions. Greenhouse conditions were short days (12 hours of light), 28°C during the day, 22°C at night, and a relative humidity of 70%.
按照与T1代相同的评估程序，对4个T1事件在T2代中进行了进一步的评估，但是每个事件采用了更多的个体。从播种期到成熟期，植物数次通过数码成像箱。在每个时间点上对每株植物从至少6个不同的角度获取数码图像(2048×1536像素，1千6百万色)。Four T1 events were further evaluated in the T2 generation following the same evaluation procedure as the T1 generation, but with more individuals per event. From sowing to maturity, the plants pass through the digital imaging box several times. Digital images (2048 x 1536 pixels, 16 million colors) were acquired from at least 6 different angles for each plant at each time point.
干旱筛选drought screening
在正常条件下在花盆土中培养来自T2种子的植物，直到进入抽穗期。然后将其转移到“干”区，停止灌溉。向随机选择的花盆中插入湿度探测仪，以监测土壤水含量(SWC)。当SWC低于一定的阈值时，自动向植物持续补水，直到再次达到正常水平。然后将植物再次重新转移到正常条件下。其余的栽培(植物成熟、种子收获)与不在非生物胁迫条件下培养的植物相同。如针对在正常条件下生长所详述的那样，记录生长和产量参数。Plants from T2 seeds were grown in potting soil under normal conditions until heading. Then move it to a "dry" area and stop watering. Moisture probes were inserted into randomly selected pots to monitor soil water content (SWC). When the SWC falls below a certain threshold, the plant is automatically replenished with water until it reaches normal levels again. The plants were then retransferred again to normal conditions. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress conditions. Growth and yield parameters were recorded as detailed for growth under normal conditions.
氮利用效率筛选Nitrogen use efficiency screening
在除营养液以外为正常的条件下在花盆土中培养来自T2种子的稻植物。从植物移植到成熟，用特定的营养液对花盆进行灌溉，所述营养液含有减小的氮(N)含量，通常少7到8倍。其余的栽培(植物成熟、种子收获)与不在非生物胁迫条件下培养的植物相同。如对正常条件下生长所详细描述的那样，记录生长和产量参数。Rice plants from T2 seeds were grown in potting soil under normal conditions except for the nutrient solution. From plant transplantation to maturity, the pots are irrigated with a specific nutrient solution containing reduced nitrogen (N) content, usually 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress conditions. Growth and yield parameters were recorded as detailed for growth under normal conditions.
盐胁迫筛选Salt Stress Screening
植物生长在由椰壳纤维和argex(3∶1)制成的基质上。在小植株移植到温室后的头两周期间应用正常营养液。过了头两周之后，向营养液中添加25mM盐(NaCl)，直至收获植物。然后测量种子相关参数。Plants were grown on a substrate made of coir and argex (3:1). A normal nutrient solution was applied during the first two weeks after transplanting the plantlets into the greenhouse. After the first two weeks, 25 mM salt (NaCl) was added to the nutrient solution until the plants were harvested. The seed-related parameters are then measured.
10.2统计学分析：F检验10.2 Statistical analysis: F test
利用双因素ANOVA(方差分析)作为统计模型，对植物表型特征进行总体评估。对用本发明基因转化的所有事件的所有植株的所有测量参数进行了F检验。进行F检验以检查基因在所有转化事件上的效应，并检验基因的总体效应，亦称为“整体基因效应”。真实整体基因效应的显著性阈值设置为F检验的5％概率水平。显著性F检验值指示存在基因效应，这意味着引起表型上差异的不仅仅是基因的存在或位置。An overall assessment of plant phenotypic characteristics was performed using two-way ANOVA (Analysis of Variance) as the statistical model. F-tests were performed on all measured parameters of all plants from all events transformed with the gene of the present invention. The F-test is performed to examine the effect of a gene on all transformation events and to examine the overall effect of a gene, also known as the "global gene effect". The significance threshold for the true overall gene effect was set at the 5% probability level of the F-test. A significant F-test value indicates the presence of a gene effect, meaning that it is not only the presence or position of the gene that is causing the difference in phenotype.
因为进行了重叠事件的两个实验，所以进行组合分析。这可以用于检查效应在两个实验中的一致性，并且如果确是如此的话，积累来自两个实验的证据以增加结论的可靠性。所使用的方法是考虑数据的多层次结构(即实验-事件-分离子)的混合模型法。通过将似然比检验与卡方分布相比较来获得P值。Because two experiments with overlapping events were performed, a combined analysis was performed. This can be used to check the consistency of the effect across two experiments and, if so, to accumulate evidence from both experiments to increase the reliability of the conclusion. The method used was a mixed modeling approach that considered the multi-level structure of the data (ie experiment-event-segregant). P values were obtained by comparing the likelihood ratio test with the chi-square distribution.
10.3测量的参数10.3 Measured parameters
生物量相关参数测量Measurement of biomass related parameters
从播种期到成熟期，植物数次通过数码成像箱。在每个时间点上对每株植物从至少6个不同的角度获取数码图像(2048×1536像素，1千6百万色)。From sowing to maturity, the plants pass through the digital imaging box several times. Digital images (2048 x 1536 pixels, 16 million colors) were acquired from at least 6 different angles for each plant at each time point.
植物地上面积(或者说叶生物量)通过计数数码图像中区别于背景的地上植物部分的像素总数而确定。此值取同一时间点从不同的角度拍摄的照片的平均值，并通过校准转换为以平方毫米表示的物理表面值。实验表明通过这种方法测量的地上植物面积与植物地上部分的生物量相关。该地上面积是在植物达到其最大叶生物量的时间点测量的面积。早期活力是萌发后三周的植物(幼苗)地上面积。根生物量增加表达为根总生物量(测量为在植物一生中观察到的最大根生物量)的增加；或者表达为根/枝条指数(测量为在根和枝条活跃生长期中根生物量和枝条生物量之间的比值)的增加。Plant aboveground area (or leaf biomass) was determined by counting the total number of pixels in the digital image that distinguish the aboveground plant parts from the background. This value is averaged over photos taken from different angles at the same time point and converted to a physical surface value expressed in square millimeters by calibration. Experiments have shown that the aboveground plant area measured by this method correlates with the biomass of the plant's aboveground parts. The aboveground area is the area measured at the point in time when the plant reaches its maximum leaf biomass. Early vigor is the aboveground area of the plant (seedling) three weeks after germination. Root biomass increase is expressed as an increase in total root biomass (measured as the maximum root biomass observed over the lifetime of the plant); or as a root/shoot index (measured as root biomass and shoot The ratio between the biomass) increases.
通过计数区别于背景的地上植物部分的像素总数，测定了早期活力。此值取同一时间点从不同的角度拍摄的照片的平均值，并通过校准转换为以平方毫米表示的物理表面值。下面描述的结果是针对萌发后3周的植物的。Early vigor was determined by counting the total number of pixels of aboveground plant parts that were distinct from the background. This value is averaged over photos taken from different angles at the same time point and converted to a physical surface value expressed in square millimeters by calibration. The results described below are for plants 3 weeks after germination.
WO 2007/093444中描述了对开花的开始的确定。Determination of the onset of flowering is described in WO 2007/093444.
种子相关参数测量Seed related parameter measurement
收获成熟的一级圆锥花序(primary panicles)、计数、装袋、贴上条形码标记，然后在烤箱中于37℃干燥三天。随后使圆锥花序脱粒，收集并计数所有的种子。使用鼓风装置使饱满谷壳和空壳分开。弃去空壳，再次计数剩下的部分。在分析天平上称重饱满的谷壳。通过计数在分离步骤之后剩下的饱满谷壳数，确定饱满种子数。通过称重从一株植物收获的所有饱满谷壳来测量每株植物的种子总重量。通过计数从植物收获的谷壳数来测量每株植物的种子总数。根据计数的饱满种子数及其总重量外推得出千粒重(TKW)。收获指数(HI)在本发明中定义为每株植物的种子总重量和地上面积(mm2)之间的比值再乘以因子106。每圆锥花序的花总数在本发明中定义为种子总数与成熟一级圆锥花序数之间的比率。种子饱满率在本发明中定义为饱满种子数占种子(或小花)总数的比例(以％表示)。Mature primary panicles were harvested, counted, bagged, barcoded, and dried in an oven at 37°C for three days. The panicles were then threshed and all seeds collected and counted. The full chaff is separated from the empty chaff using an air blower. Discard the empty shells and count the remainder again. Weigh the full chaff on an analytical balance. The number of filled seeds was determined by counting the number of filled chaffs remaining after the separation step. The total seed weight per plant was measured by weighing all plumped chaff harvested from a plant. Measure the total number of seeds per plant by counting the number of husks harvested from the plant. The thousand-kernel weight (TKW) was extrapolated from the number of filled seeds counted and their total weight. The Harvest Index (HI) is defined in the present invention as the ratio between the total seed weight and the aboveground area (mm2 ) per plant multiplied by a factor of 106 . The total number of flowers per panicle is defined in the present invention as the ratio between the total number of seeds and the number of mature primary panicles. The seed filling rate is defined in the present invention as the ratio (expressed in %) of the number of full seeds to the total number of seeds (or florets).
实施例11：转基因植物的表型评估结果Example 11: Results of Phenotypic Evaluation of Transgenic Plants
11.1.Brevis Radix样(BRXL)多肽11.1. Brevis Radix-like (BRXL) polypeptides
在组成型启动子的控制下表达编码SEQ ID NO：18所示的BRXL多肽的核酸序列的T1代转基因稻植物的评估结果在下文表D中呈现。The results of the evaluation of T1 generation transgenic rice plants expressing the nucleic acid sequence encoding the BRXL polypeptide set forth in SEQ ID NO: 18 under the control of a constitutive promoter are presented in Table D below.
与对照相比较，转基因植物的植物高度和千粒重(TKW)显著增加。The plant height and thousand-kernel weight (TKW) of the transgenic plants were significantly increased compared to the controls.
表D1：在用于组成型表达的启动子控制下表达编码SEQ ID NO：18所示的BRXL多肽的核酸序列的T1代转基因稻植物的评估结果。Table D1: Evaluation results of T1 generation transgenic rice plants expressing the nucleic acid sequence encoding the BRXL polypeptide shown in SEQ ID NO: 18 under the control of the promoter for constitutive expression.
11.2.ARP6多肽11.2. ARP6 polypeptide
在非胁迫条件下表达包含SEQ ID NO：101中的最长开放阅读框的核酸的T2代转基因稻植物的评估结果在下文中呈现。关于转基因植物的产生的详细内容参见之前的实施例。The results of the evaluation of T2 generation transgenic rice plants expressing the nucleic acid comprising the longest open reading frame in SEQ ID NO: 101 under non-stress conditions are presented below. See previous examples for details on the generation of transgenic plants.
在非胁迫条件下的转基因稻植物的评估结果示于下文中。对于种子总产量(totalwgseeds)、饱满种子数(nrfilledseed)、饱满率(fillrate)和收获指数(harvestindex)观察到了至少5％的增加。The results of the evaluation of transgenic rice plants under non-stress conditions are shown below. An increase of at least 5% was observed for total seed yield (totalwgseeds), number of filled seeds (nrfilledseed), filling rate (fillrate) and harvest index (harvestindex).
表D2：Form D2:
11.3.POP多肽11.3. POP polypeptides
在非胁迫条件下表达编码SEQ ID NO：117的POP多肽的核酸的T2代转基因稻植物的评估结果在下列表D3中呈现。当在非胁迫条件下生长时，对于地上生物量(AreaMax)、根生物量(RootMax和RootThickMax)和对于种子产量(种子总重量、饱满种子数、饱满率、收获指数)，观察到了至少5％的增加。此外，表达POP核酸的植物显示出了更快的生长速率(从播种至植物达到其最终生物量的90％的日期，需要更短的时间(以天表示)(AreaCycle)；以及更早的开花开始(TimetoFlower：播种至第1个圆锥花序出现之间的时间(以天表示))。The results of the evaluation of T2 generation transgenic rice plants expressing a nucleic acid encoding the POP polypeptide of SEQ ID NO: 117 under non-stress conditions are presented in Table D3 below. When grown under non-stress conditions, at least 5% was observed for aboveground biomass (AreaMax), root biomass (RootMax and RootThickMax) and for seed yield (total seed weight, number of filled seeds, filling rate, harvest index) increase. In addition, plants expressing the POP nucleic acid showed a faster growth rate (required a shorter time (expressed in days) from sowing to the date at which the plant reached 90% of its final biomass (AreaCycle); and earlier flowering start(TimetoFlower: time (in days) between sowing and first panicle appearance).
表D3：转基因稻植物的数据总结；对于每一个参数，针对确认(T2代)，显示总体的增加百分比，对于每一个参数，p值＜0.05。Table D3: Summary of data for transgenic rice plants; for each parameter, for validation (T2 generation), the overall percentage increase is shown, for each parameter, p-value < 0.05.
11.4.Crumpled Leaf(CRL)多肽11.4. Crumpled Leaf (CRL) polypeptide
在非胁迫条件下评估的、表达包含SEQ ID NO：155中的最长开放阅读框的核酸的T2代转基因稻植物的、评估结果在下文中呈现。关于转基因植物的产生的详细内容参见之前的实施例。The results of the evaluation of T2 generation transgenic rice plants expressing a nucleic acid comprising the longest open reading frame in SEQ ID NO: 155 evaluated under non-stress conditions are presented below. See previous examples for details on the generation of transgenic plants.
在非胁迫条件下转基因稻植物的评估结果在下文中呈现。对于种子的总产量(Totalwgseeds)、饱满种子数(nrfilledseed)、饱满率(fillrate)和收获指数(harvestindex)，观察到了至少5％的增加。The results of the evaluation of transgenic rice plants under non-stress conditions are presented below. An increase of at least 5% was observed for total seed yield (Totalwgseeds), number of filled seeds (nrfilledseed), filling rate (fillrate) and harvest index (harvestindex).
表D4.Table D4.
Claims (28)
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| PCT/EP2009/065906WO2010063637A1 (en) | 2008-12-03 | 2009-11-26 | Plants having enhanced abiotic stress tolerance and/or enhanced yield-related traits and a method for making the same |
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